Note: Within nine months of the publication of the mention of the grant of the European patent in the European PatentBulletin, any person may give notice to the European Patent Office of opposition to that patent, in accordance with theImplementing Regulations. Notice of opposition shall not be deemed to have been filed until the opposition fee has beenpaid. (Art. 99(1) European Patent Convention).

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(12) EUROPEAN PATENT SPECIFICATION

(45) Date of publication and mentionof the grant of the patent:09.06.2010 Bulletin 2010/23

(21) Application number: 03719654.0

(22) Date of filing: 09.04.2003

(51) Int Cl.:C12N 9/04 (2006.01) C12Q 1/00 (2006.01)

C12Q 1/02 (2006.01) C12Q 1/04 (2006.01)

C12Q 1/26 (2006.01) C12Q 1/32 (2006.01)

G01N 33/50 (2006.01)

(86) International application number:PCT/US2003/010872

(87) International publication number:WO 2003/089635 (30.10.2003 Gazette 2003/44)

(54) CYTOTOXICITY ASSAY

ZYTOTOXIZITÄTSTEST

ESSAI DE CYTOTOXICITE

(84) Designated Contracting States:AT BE BG CH CY CZ DE DK EE ES FI FR GB GRHU IE IT LI LU MC NL PT RO SE SI SK TR

(30) Priority: 17.04.2002 US 124029

(43) Date of publication of application:26.01.2005 Bulletin 2005/04

(73) Proprietor: PROMEGA CORPORATIONMadison, Wisconsin 53711 (US)

(72) Inventors:• RISS, Terry, L.

Oregon, WI 53575 (US)• MORAVEC, Richard, A.

Oregon, WI 53575 (US)

(74) Representative: Tollett, Ian et alWilliams PowellStaple Court11 Staple Inn BuildingsLondon, WC1V 7QH (GB)

(56) References cited:US-A- 4 849 347 US-A- 5 501 959US-A- 5 912 139 US-B1- 6 811 990

• O’BRIEN J.: ’Investigation of the alamar blue(resazurin) fluorescent dye for the assessment ofmammalian cell cytotoxicity’ EUROPEAN J.BIOCHEMISTRY vol. 267, no. 1, 2000, pages 5421- 5426, XP002968559

• RASMUSSEN E.: ’Use of fluorescent redoxindicators to evaluate cell proliferation andviability’ IN VITRO AND MOLECULARTOXICOLOGY vol. 12, no. 1, 1999, pages 47 - 58,XP002968560

• MCMILLAN M.K.: ’An improved resazurin basedcytotoxicity assay for hepatitic cells’ CELLBIOLOGY AND TOXICOLOGY vol. 18, no. 3, 2002,pages 157 - 173, XP002968561

• ANONYMOUS: "Cytotox 96 Test" PROMEGATB163, 2001,

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Description

FIELD OF THE INVENTION

[0001] The invention is directed to assays for deter-mining the cytotoxic effect of a given test compound ora given set of test conditions. The inventive methodmeasures the release of a cytoplasmic component fromdead and dying cells, the cytoplasmic component havinga half-life in culture medium greater than two hours, andpreferably greater than four hours at 37˚C.The cytoplasmic component is measured by the conver-sion of an indicator dye. The preferred cytoplasmic com-ponent to be measured is lactate dehydrogenase (LDH),an enzyme that catalyzes the oxidation of lactate to pyru-vate. Thus, in the preferred embodiment, an LDH-cata-lyzed oxidation of lactate is linked to a diaphorase-cata-lyzed reduction of a non-fluorescent species to yield afluorophore. Kits for practicing the invention are also dis-closed.

BACKGROUND

[0002] Methods to determine cell viability or cytotoxic-ity in response to exposure to a given test agent are keyto pharmaceutical and environmental testing, pesticideand herbicide testing, drug discovery, etc. In short, todetermine whether a given chemical agent presents areal or potential risk when exposed to a given cell typerequires a method that reliably, precisely, and accuratelymeasures cell toxicity and/or viability after exposure tothe test agent.[0003] A common method of determining cell viabilityis based on the ability ofthe membrane of viable cells toexclude vital dyes such as trypan blue and propidiumiodide. Living cells exclude such vital dyes and do notbecome stained. In contrast, dead or dying cells that havelost membrane integrity allow these dyes to enter thecytoplasm, where the dyes stain various compounds ororganelles within the cell.[0004] Non-viable cells that have lost membrane in-tegrity also leak cytoplasmic components into the sur-rounding medium. Cell death thus can be measured bymonitoring the concentration of these cellular compo-nents in the surrounding medium. One such method isdescribed in Corey et al. (1997) J. Immunol. Meth. 207:43-51. In this assay, the release of glycerldehyde-3-phosphate dehydrogenase (G3PDH) from dead or dam-aged cells is measured by coupling the activity of thereleased G3PDH to the production of ATP.[0005] Other methods to test for cell viability or celldeath rely upon the conversion of a dye from one stateto another. For example, in a typical format, prior to thereaction the dye absorbs at a first wavelength of radiation.The dye is then converted to a product that absorbs at asecond (and different) wavelength of light. By monitoringthe conversion ofthe dye from one state to the other, theextent of cell viability or cell death can be determined. A

number of suitable dyes for this purpose are known inthe art. The most frequently used of these indicators areelectron-acceptor dyes such as tetrazolium salts. Tetra-zolium salts known in the prior art include MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide),XTT (sodium 3’-{(1-phenylamino-carbonyl)-3,4-tetrazo-lium}-bis(4-methoxy-6-nitro)benzenesulfonic acid hy-drate), and MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-car-boxymethoxyphenyl)- 2-(4- sulphophenyl)- 2H- tetrazo-lium, inner salt).[0006] A typical cell viability and proliferation assay us-ing MTS has been described (Buttke et al., (1993) J. Im-munol Methods, 157: 233-240). Dunigan et al. (1995,BioTechniques, 19:640-649) proceed to describe thatone of the hallmarks of metabolism is the generation ofenergy via complex redox reactions of organic mole-cules. A great many of these reactions utilize β-nicotina-mide adenine dinucleotide (NADH) or β-nicotinamide ad-enine dinucleotide phosphate (NADPH) as hydrogen do-nors. While it is theoretically possible to monitor NADHand NADPH concentrations directly via spectrophotom-etry, from a practical standpoint, direct spectrophotomet-ric analysis is limited due to the presence numerous com-ponents that absorb light near the absorption maximumof NADH and NADPH (ε = 16,900 at λmax of 259 nm).For example, NAD+, NADP+, DNA, RNA, and most pro-teins have absorption maxima at approximately 260 nm.[0007] Buttke et al. describe using MTS to measureindirectly the reduction caused by living, proliferatingcells, MTS having an absorbance in the visible regionwhen in its reduced form. The reduced, formazen formof MTS is water soluble and has a broad absorption max-imum centered at 450-580nm. The experiments de-scribed by Dunigan et al. are entirely cell free. MTS andMTS/phenazine methosulfate (PMS) solutions were pre-pared as stock solutions and various combinations ofenzymes and reducing agents were added to aliquots ofthe stock solutions and analyzed spectrophotometricallyover time. Various combinations of NADH, NADPH, dithi-othreitol, 2-mercaptoethanol, malic acid, isocitric acid,malate dehydrogenase, and isocitrate dehydrogenasewere tested. The authors found that MTS alone convertsonly very slowly to its reduced formazen structure. Re-activity of the MTS, however, is hugely accelerated byadding 5% of the electron transfer reagent PMS to thereaction solution. Thus, the authors conclude thatMTS/PMS is a useful monitor of NADH and NADPH gen-eration in cell-free aqueous systems.[0008] Lancaster et al., U.S. Patent No. 5,501,959, is-sued 26 March 1996, describe a cell viability and prolif-eration assay wherein microorganisms, tissue cells, orthe like, are incubated in a growth medium in the pres-ence of the dye resazurin and a compound to be tested.A redox stabilizing agent, dubbed a "poising" agent, isalso added to the reaction mix to inhibit non-specific au-toreduction of the resazurin due to components foundwithin most culture media. In this assay, the resazurindye is reduced by the activity of living cells. Thus, in the

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Lancaster et al. assay, the resazurin dye is used as aredox indicator to detect microbial growth, not microbialdeath. The reduced form of resazurin, known trivially asresorufin, can be detected fluorimetrically or colorimetri-cally. Resazurin, the oxidized form of the dye, is blue,while resorufin, the reduced form of the dye, is red.[0009] Several cytotoxicity assays can be purchasedcommercially. For example, Molecular Probes of Eu-gene, Oregon, markets a cytotoxicity assay kit under thetrademark "Vybrant". The "Vybrant"-brand assay detectsthe release of the cytoplasmic enzyme glucose-6-phos-phate dehydrogenase (G6PDH) from dead and dyingcells. This assay detects G6PDH via a two-step processthat leads to the reduction of resazurin to resorufin. Mo-lecular Probes’ product literature specifically states thatincubations longer than 24 hours "will result in significantdegradation of G6PDH, impairing the assay results". SeeMolecular Probes’ product information flier no. V-23111,revised 22 October 2001. In the hands of the presentinventors, however, the half-life of the G6PDH followingcell lysis at 37˚C was estimated to be less than two hoursat 37˚C, thus rendering this kit unsuitable for cytotoxictesting over longer spans of time.[0010] Promega Corporation of Madison, Wisconsin,markets a line of cell viability, cytotoxicity, and cell pro-liferation assays under the trademarks "CellTiter 96",CellTiter-Glo", and "CytoTox 96". See Promega Techni-cal Bulletin Nos. 112 (published 1991), 169 (publishedJune 1993), 245 (published August 1996), and 288 (pub-lished March 2001).[0011] The product literature "CytoTox 96, Non-Radi-oactive Cytotoxicity Assay," (Promega CorporationTechnical Bulletin 163, published September 1992) de-scribes a colorimetric assay that can be used in place ofCr-51 release cytotoxicity assays. This assay measuresquantitatively the release of lactate dehydrogenase uponcell lysis. The LDH in culture supernatants is measuredwith a coupled enzymatic assay which results in the con-version of a tetrazolium salt into a red formazan product.The amount of color formed is proportional to the numberof lysed cells.[0012] Promega’s "CytoTox 96"-brand non-radioac-tive cytotoxicity assay, for example, is a colorimetric as-say for determining the cytotoxicity of a test compound.This assay quantitatively measures the release of LDHfrom dead cells using an enzyme-linked reduction of INT,and MTS-like tetrazolium dye (see U.S. Patent No.5,185,450 for a full description of the MTS dye). Thisassay is a two-step protocol (i.e., it is non-homogeneous)and the INT dye is detected colorimetrically. This assayuses conditions that are incompatible with living eukary-otic cells as the reaction takes place at a pH of 8.5 witha detergent (Triton-X100) present.[0013] Genotech of St. Louis, Missouri, markets a lac-tate dehydrogenase (LDH)-based cytotoxicity assay un-der the trademark "CytoScan". The "CytoScan"-brandassay is a colorimetric method that measures LDH re-leased from dead cells. The LDH released by dead cells

is measured via a coupled enzymatic reaction that resultsin the reduction of a tetrazolium salt into a red-coloredformazen. The LDH activity is then determined as a func-tion of NADH oxidation or tetrazolium reduction over adefined period of time. See Genotech catalog no.786-210. Essentially identical assay kits are marketedby Panvera of Madison, Wisconsin (LDH Cytotoxicity De-tection Kit, Panvera product no. TAK MK401), OxfordBiomedical Research of Oxford, Michigan (ColorimetricCytoxicity Assay Kit, Oxford product no. LK 100), andRoche Molecular Biochemicals of Indianapolis, Indiana(Cytotoxicity Detection Kit, Roche catalog no. 1 644 793).All of these kits require a two step procedure to removeculture medium to a separate container and thus are non-homogeneous.[0014] Sigma of St. Louis, Missouri, markets two dif-ferent in vitro toxicology assay kits under the ProductNames "Tox-7" and "Tox-8". The "Tox-7" kit is LDH-based and is essentially identical to Genotech’s "Cyto-Scan"-brand assay described in the preceding para-graph. The "Tox-7" assay is a two-step process that re-quires transferring a supernatant or.a cell lysate to a sep-arate vessel, where the supernatant or lysate is then an-alyzed. Two-step processes are not preferred for high-throughput screening due to the increased material han-dling requirements. The particular tetrazolium dye usedin Sigma’s "Tox-T’ kit is not specified, but the productliterature indicates that the reaction is measured spec-trophotometrically at 490 nm, the absorption maximumtypical for formazans (and the same wavelength speci-fied in the Genotech product).[0015] All of these commercially available kits requiretransfer of culture supernatant to an additional vessel forenzymatic measurement of LDH activity and are there-fore non-homogeneous.[0016] In Sigma’s "Tox-8" assay, bioreduction of resa-zurin by viable cells (not dead cells) results in the forma-tion of resorufin. The amount of dye reduced can bemeasured fluorimetrically or spectrophotometrically.[0017] BioSource, International (Camarillo, California)markets kits for measuring cell proliferation and viabilityunder the trademark "alamarBlue," (see catalog nos.DAL1025 and DAL1100). Like resazurin, "alamarBlue"-brand dye can be used to monitor the reducing environ-ment of living cells. The technology underlying this com-mercial product is described in Lancaster, U.S. PatentNumber 5,501,959.[0018] The use of absorbent pads impregnated with re-sazurin and antibiotics for antimicrobial susceptibility test-ing are described in Baker et al. (1980) Microbiol. 26:248-253 and Canadian Patent No. 1,112,140. Bacterialisolates are applied to the pad in a brain heart infusionbroth. The protocols described, however, are not suitablefor determining minimum inhibitory concentrations (MIC).Kanazawa et al. (1966) J. Antibiotics 19:229-233 also de-scribe the use of absorbent pads impregnated with resa-zurin and antimicrobial agents for use in susceptibility test-ing. Brown et al. (1961) J. Clin. Path. 5:10-13 and U.S.

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Patent No. 3,107,204 describe the use of absorbent padsimpregnated with a tetrazolium redox indicator and anti-microbial agents, also for use in susceptibility testing.[0019] There remains, however, a long-felt and unmetneed for a cytotoxicity assay that measures cell death(rather than viability), wherein the assay is rapid, the com-ponents are non-toxic (and thus the test can be run inthe presence of the cells being investigated), and whereinthe cytoplasmic components measured have a half-lifegreater than about two hours.

SUMMARY OF THE INVENTION

[0020] In recognition of the above-described short-comings, a first embodiment of the invention is directedto a method for determining the cytotoxicity of a testagent. The method comprises first adding a pre-deter-mined amount of the test agent to a vessel containingliving cells in culture medium. The cells are then incubat-ed in the culture medium for a pre-determined amountof time. To the incubated cells and culture medium isthen added a reagent mixture that is non-toxic to the still-living cells in the culture medium. The reagent mixturecomprises a solvent, a dye having an oxidized state anda reduced state wherein the reduced state can be distin-guished from the oxidized state and wherein the dye isinitially present in the oxidized state, an electron transferagent, a substrate for a cytoplasmic enzyme having ahalf-life greater than two hours and a cofactor for thecytoplasmic enzyme. The contents of the vessel are thenmeasured for production of the reduced state of the dye.The reduced state of the dye is produced by a cytoplas-mic enzyme that will convert the substrate included inthe reagent mixture to the desired product. The cytoplas-mic enzyme is released from dead and dying cells withinthe vessel, but may also be present as a component inthe cell medium such as serum. Thus, by quantifying theamount of this enzyme indirectly (by detecting the re-duced state of the dye) and subtracting backgroundamounts present before cell compromise takes place,cell death caused by the test agent is determined.[0021] The invention also encompasses kits to carryout the above-described method. The reagent mixturethat is used in the method (and provided in the kit) canbe added directly to the culture medium (with the cellspresent) without adversely affecting cell viability for theduration of the assay. In the preferred embodiment, themethod and the corresponding kit yield results essentiallyimmediately, generally in about 10 minutes. Data can begathered either colorimetrically or fluorimetrically. Fluor-ometric measurement is much more sensitive and is thepreferred means of resorufin detection. In other embod-iments, data can be collected very quickly after additionof the reagent mixture, with the data gathering processgenerally being possible in a matter of seconds or min-utes.[0022] It is much preferred that the entire method beconducted in a single vessel. That is, the cells are placed

into a vessel containing suitable medium. The test agentis added to the vessel. The vessel with the cells and thetest agent is incubated. The reagent mixture is then add-ed to the same vessel, in the presence of the incubatedcells (rather than taking a cell-free aliquot and adding thereagent mixture to it). The cell medium is then measured(colorimetrically or fluorimetrically). The preferred em-bodiment of the invention is thus "homogeneous," homo-geneous designating that the entire method is conductedin a single vessel, rather than requiring aliquots to betransferred and tested in a different vessel. This makesthe present method very highly suitable for high-through-put automation, where it is desirable that material han-dling and manipulation requirements be reduced to anabsolute minimum.[0023] As noted above, the output from the methodbegins to occur essentially immediately after adding thereagent mixture to the incubated cells. Thus, data gath-ering can begin promptly after adding the reagent mix-ture. The signal generated by the method may be meas-ured for anywhere from a matter of seconds, to ten min-utes, to thirty minutes or longer. In the preferred embod-iment, the signal is measured following a period of timegenerally on the order of 10 minutes.[0024] In the preferred embodiment of the method, thesubstrate for a cytoplasmic enzyme having a half-lifegreater than two hours is lactate, and the cytoplasmicenzyme that forms the basis of the cytotoxic measure-ment is lactate dehydrogenase (LDH). LDH has a half-life of approximately 9 hours in culture medium at 37˚Cfollowing cell lysis. The preferred dye for use in the in-vention is resazurin. MTS may also be used. The pre-ferred electron transfer agent is the enzyme diaphorase,although Meldola’s Blue also may be used.[0025] It is also preferred that a stop solution (prefer-ably a solution of sodium dodecylsulfate, SDS) be addedto the reaction prior to measuring for production of thereduced state of the dye.[0026] The present method relies upon cytoplasmiccomponents that have a long half-life after release fromdead and dying cells. Thus, as noted above, in the pre-ferred embodiment, LDH is the cytoplasmic componentmeasured. Because the method utilizes a cytoplasmiccomponent with a relatively long half-life, minimal deg-radation of the component will occur over a test periodthat may include incubation times of 12 or 24 hours (orlonger). The activity of the released cytoplasmic compo-nent with a long half-life can be measured more reliablythan a released component with a short half-life, espe-cially when the incubation period of the cells with the testagent results in release of the cytoplasmic componentseveral hours before the reagent mixture is added to thevessel.[0027] It is much preferred, although not required, thatthe method be performed in a "homogenous" fashion,meaning that all of the steps of the method are carriedout in the same vessel, in the presence of the cells beingused in the method. This can be done, for example, in a

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test tube, a multi-well plate, or any other suitable vessel.[0028] A second embodiment of the invention is direct-ed to a kit for carrying out the above-described method.The kit comprises, in combination, a dye disposed in afirst container, the dye having an oxidized state and areduced state wherein the reduced state can be distin-guished from the oxidized state and wherein the dye ispresent in the oxidized state; a lyophilized substrate mixdisposed in a second container, the substrate mix com-prising an electron transfer agent, a substrate for a cy-toplasmic enzyme having a half-life greater than twohours, and a cofactor for the cytoplasmic enzyme; and(optionally) instructions for use of the kit.[0029] In the preferred embodiment of the kit, the re-agent mix is an aqueous solution comprising: about 109mM lactate; about 3.3 mM NAD+; about 2.2 U/ml diapho-rase about 3.0 mM Tris buffer; about 30 mMHEPES;about 10 mM NaCl; and about 350 mM resazurin. Othercomponent concentrations may be used to the extentthat the substrate, the cofactor and the electron transportagent are present in excess so that there is a suitablerate of reaction and to the extent that the osmolality ofthe reagent mix does not adversely affect the overall os-molality of the culture medium to the point that the cellsare damaged.[0030] Optionally, a stop solution may be disposed ina third container. The stop solution generally comprisesa soap, a detergent, or a strong base. Preferred stopsolutions include SDS or sodium hydroxide.[0031] Also optionally, a lysis solution (for positive con-trol experiments) may be disposed in an additional(fourth) container. Preferred lysis solutions include 9%w/v Triton X-100 in water.[0032] Another embodiment of the kit may contain areagent mixture in a single container. Such kits comprise,in combination, a buffer, a dye, a substrate for the cyto-plasmic component, an appropriate cofactor for the cy-toplasmic component, and an electron transfer agent, alldisposed in a first container. For example, such a firstcontainer would contain, in combination, resazurin, lac-tate, NAD+, diaphorase, Tris, HEPES and NaCl. The kitalso contains instructions for the kit, and optionally, astop solution.[0033] A primary advantage of the preferred embodi-ment of the present invention is that it provides improvedmedia and methods that employ resazurin or MTS as anindicator of cell death. In particular, the inventive methodutilizes a reagent mixture that is not deleterious to theviability of the cells being tested. Thus, the inventivemethod is "homogeneous" in that it can be performed inthe same vessel in which the cells are incubated, withoutthe need to remove a cell-free aliquots of the culture me-dium. In short, using the present invention, there is noneed to draw a cell-free aliquot of the cell culture mediumand run the test on the aliquot. The method can be per-formed directly upon the cell medium, in the presence ofthe incubated cells; in the same vessel in which the cellswere incubated.

[0034] Another advantage of the invention is that, inthe preferred embodiment, it measures the activity ofLDH to determine cell death. Because LDH has a longhalf-life (greater than 8 hours at 37˚C), the invention canbe used to determine the extent of cell death over a longtesting period, more accurately and more precisely thanprior art methods.[0035] Another advantage of the invention is that be-cause the assay conditions are not deleterious to thecells being tested, the living cells present in the culturewhen the assay is performed generate no more than aninsignificant amount of signal above the background.This is because the cell membranes of the viable cellsare not damaged by the reagent solution used in themethod, and thus these viable cells retain their cytoplas-mic LDH. Therefore, only LDH present in the medium,from a medium additive such as serum or released frommembrane-compromised cells, is measured. This allowsthe assay to be performed directly in vessels containingviable cells.[0036] The utility of the assay is manifest in that deter-mining the cytotoxicity of a given compound or compo-sition is a critical piece of information in the drug discoveryprocess. For example, cytotoxicity is desirable when thetoxicity is specifically displayed against an identified tar-get cell type, such as a given type of cancer, an infectious,disease-causing microorganism, or a parasitic organism.Cytotoxicity, of course, is undesirable when, for example,a putative new drug is discovered to be cytotoxic to nor-mal cells. The subject invention can be used in both in-stances to measure the cytotoxicity of a given test agentagainst a given cell type.

BRIEF DESCRIPTION OF THE DRAWINGS

[0037]

FIG. 1 is a schematic rendering of the preferredmethod according to the present invention.FIG. 2 is a graph depicting cell number (X-axis) ver-sus the fluorescence (560nm excitation and 590nmemission (Y-axis) for an exemplary run of the presentmethod. Thetest was performed using L929cells(ATCC CCL-1). Intact cells (O), lysed cells (s).FIG 3 is a graph depicting time after start of lysis inhours (X-axis) versus the fluorescence (560nm ex-citation and 590nm emission) (Y-axis) in an assaymeasuring the activity of G6PDH released fromlysed L929 cells (O) and Jurkat cells (h).FIG 4 is a graph depicting time after start of lysis inhours (X-axis) versus the fluorescence (560nm ex-citation and 590nm emission) (Y-axis) in an assaymeasuring the activity of LDH released from lysedL929 cells (M) and Jurkat cells (s).FIG. 5 is a graph depicting the cytotoxicity of TNFαas measured using the method of the present inven-tion. TNFα concentration in ng/ml is presented onthe X-axis; fluorescence is presented on the Y-axis

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as in Figures 2 through 4. See Example 3.

DETAILED DESCRIPTION OF THE INVENTION

[0038] The following abbreviations are used through-out the specification and claims. Words not expresslydefined herein are to be given their normal and acceptedmeaning in the art.[0039] "L929 cells" = a fibroblastoid adherent cell linederived from C3H/An mouse subcutaneous areolar andadipose tissue, available commercially from the Ameri-can Type Culture Collection (Manassas, Virginia) underaccession no. ATCC CCL-1.[0040] "Jurkat cells" = a human lymphocyte T-cell line,available commercially from the ATCC under accessionno. ATCC TIB-152.[0041] "HEPES" = N-2-hydroxyethylpiperazine-N’-2-ethanesulfonic acid.[0042] "Known or putative pharmaceutical or thera-peutic agent" = a chemical compound or formulationknown and used as a pharmaceutical agent, or a chem-ical compound or formulation being investigated for useas a pharmaceutical agent.[0043] "LDH" = lactate dehydrogenase.[0044] "MTS" = 3-(4,5-dimethylthiazol-2-yl)-5-(3-car-boxymethoxyphenyl)- 2-(4- sulphophenyl)- 2H- tetrazo-lium, inner salt, available commercially from PromegaCorp, Madison, Wisconsin.[0045] "MTT" = 3-(4,5-dimethylthiazol-2-yl)-2,5-diphe-nyltetrazolium bromide, available commercially fromAldrich.[0046] "NADH" = β-nicotinamide adenine dinucleotide(protonated form). "NAD+" indicates unprotonated form.[0047] "NADPH" = β-nicotinamide adenine dinucle-otide phosphate (protonated form). "NADP+" indicatesunprotonated form.[0048] "resazurin" = 7-hydroxy-3H-phenoxazin-3-one10-oxide (sodium salt), available commercially fromAldrich (catalog no. 19,930-3).[0049] "resorufin" = 7-hydroxy-3H-phenoxazin-3-one(the reduced form of resazurin), available commerciallyfrom Aldrich (catalog no. 42,445-5).[0050] "SDS" = sodium dodecylsulfate.[0051] "Tris" = tris(hydroxymethyl)aminomethane.[0052] "Vessel" = indicates any container or holderwherein the inventive method can occur. Includes singlewell containers, such as test tubes, and multi-well con-tainers such as microtiter plates of any configuration."Vessel" also encompasses pads, patches, tapes, band-ages, and the like, of any material construction, that aresufficiently absorbent to retain the cells and reagentsneeded to perform the subject method.[0053] "XTT" = sodium 3’-{(1-phenylamino-carbonyl)-3,4-tetrazolium}-bis(4-methoxy-6-nitro)benzene-sulfon-ic acid hydrate.[0054] Referring now to FIG. 1, which is a schematicdiagram of the preferred method according to the presentinvention, the method comprises incubating cells in a

growth medium and an agent to be tested (hereinafterreferred to as the test agent). The cells are then incubatedin the presence of the reagent mixture and the test agentfor a period of time. Assuming the test agent, not shownin Fig. 1, is at least partially cytotoxic, this will cause somecells to die and become "leaky", thereby releasing cyto-plasmic components, such as LDH , into the culture me-dium.[0055] The reagent mixture used in the method is non-toxic to the living cells. Thus it can be added directly tothe cells being incubated, without adversely affecting theoutcome of the method. The reagent mixture comprisesa solvent (preferably water, and preferably buffered), adye having an oxidized state and a reduced state whereinthe reduced state can be distinguished from the oxidizedstate and wherein the dye is initially present in the oxi-dized state, an electron transfer agent, a substrate for acytoplasmic component (such as lactate, a substratespecific for LDH), and an appropriate cofactor (such asNAD+ for LDH) As indicated in Fig. 1, the dye preferablyis resazurin, although the tetrazolium dye MTS can alsoused with success. Other redox indicators may also beused. The preferred electron transfer agent is the enzymediaphorase, but Meldola’s Blue (also known as naphtholblue, color index C.I.51175) (EM Science/Harleco, a di-vision ofMerck KGaA, Darmstadt, Germany) may alsobe used.[0056] In the preferred embodiment, the cells are in-cubated for a pre-determined amount of time in the pres-ence of the test agent, after which time the reagent mix-ture is added to the incubated cells. As shown in Fig. 1,when lactate is used as the substrate, and LDH is thecytoplasmic component being measured, LDH releasedfrom dead or dying cells initiates an enzyme-catalyzedseries of reactions. In the first reaction, lactate (one ofthe ingredients included in the reagent solution) is oxi-dized to form pyruvate. This reaction requires NAD+ andthus, in the preferred embodiment, NAD+ is included inthe reagent mixture.[0057] The oxidation of lactate to pyruvate results inthe production of NADH. In a second reaction, the dye(resazurin is shown in Fig. 1) is reduced in a reactionpowered by the NADH produced in the first reaction andcatalyzed by the electron transfer agent (in Fig. 1, dia-phorase, the preferred electron transfer agent, is shown).Thus, as illustrated in Fig. 1, the preferred electron trans-fer agent diaphorase catalyzes the reduction of resazurinto resorufin, a reduction powered by NADH.[0058] The end result is a method wherein the releaseof a cytoplasmic component (LDH is shown in Fig. 1)from dead and dying cells is measured indirectly bymeasuring the reduction of a dye, that reduction beingdriven via a reaction that is linked to the oxidation of asubstrate specific for the released cytoplasmic compo-nent (e.g., the lactate to pyruvate conversion caused bythe released LDH as shown in Fig. 1). The amount of thereduced dye formed is proportional to the number of lysedcells. (See the Examples below for a further discussion.)

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[0059] The production of the reduced form of the dyecan be measured colorimetrically or fluorimetrically, orother electromagnetic spectral measuring device, de-pending upon the dye. Resorufin can be measured eithercolorimetrically or fluorimetrically, however, MTS can on-ly be measured colorimetrically. Suitable measuring de-vices are exceedingly well known in the art and can bepurchased from a host of commercial suppliers.[0060] In particular, when the dye used is resazurin,reduction can be detected either by absorbance color-imetry or by fluorimetry. Resazurin is deeply blue in colorand is essentially non-fluorescent, depending upon itslevel of purity. Resorufin, the reduced form of resazurin,is red and very fluorescent. When using colorimetry, thereaction is monitored at wavelengths well known in theart to be an absorption maximum for resorufin (approxi-mately 570 nm). Fluorescence measurements of resoru-fin are made by exciting at wavelengths well known inthe art (approximately 530 to 560 nm) and measuring theemission spectrum (known to the art to have a maximumat about 590 nm). Because fluorometric detection is moresensitive than spectrophotometric detection, in the pre-ferred embodiment of the method, resazurin is used asthe dye and the production of resorufin is detected fluor-imetrically.[0061] The medium in which the cells are grown orheld does not limit the functionality ofthe invention, al-though non-reducing media are preferred to minimizeany non-specific reduction of the dye. For microbial cul-tures, suitable media include, without limitation, Mueller-Hinton Broth and trypticase soy broth. For mammaliancell cultures, suitable media include, without limitation,RPMI 1640, RPMI 1640 plus fetal bovine serum, andDulbecco’s Modified Eagle Medium, Hanks’ BalancedSalt Solution, Phosphate Buffered Solution. It has beenobserved that media naturally containing or supplement-ed with sodium pyruvate slows the rate of the LDH reac-tion and thus the rate of resorufin generation. Dependingupon desired sensitivity, longer development times maybe desired.[0062] In one application of the method, the test agentwill be a putative growth-inhibiting substance, such asan antimicrobial agent or a cytotoxic drug. The nature ofthe test agent, however, is virtually unlimited. The testagent could be, for example, any element, compound,mixture, drug, or putative drug, in any form (such as solid,liquid, or gas) or a set of environmental conditions (suchas any combination of heat, humidity, light, oxygen level,and the like) desired to be tested for its cytotoxic effects.The method can also be used to test known antimicrobialagents and cytotoxic drugs for the purpose of determiningwhich chemotherapeutic agents would be most effectiveagainst a given infection or cell type. The method canalso be utilized with unknown or suspected antimicrobialagents and drugs for the purposes of determining theirpotential activity against a given microorganism or celltype, e.g., high-throughput screening of substances forbiological activity as part of drug screening or other ac-

tivities.[0063] Cells to be used in the method may be collectedfrom any source, and may be eukaryotic or prokaryotic.For example, human cells may be collected by doctorsin their offices and sent to a central testing laboratory fortesting using the present method, or cell specimens maybe collected from patients in a hospital. The microorgan-ism specimens may come from any part of the human oranimal body, such as from cerebral spinal fluid, an ab-scess, an infected wound, genital infections, etc. Thecells may be from tumor biopsies or other specimens.The cells to be tested may also come from food samples,soil samples, etc. In short, the nature of the cells to betested and their source is not critical to the invention.[0064] The collected specimens are cultured on or ina suitable medium, as noted above, in accordance withconventional laboratory practice. From the bacterial col-onies or cellular clones on the primary culture plate, aninoculum is prepared in accordance with an establishedprocedure which produces a bacterial or cellular suspen-sion of a prearranged concentration. Further processingof the suspension depends on the particular apparatusand method to be used for susceptibility testing.[0065] One preferred outcome of bacterial testing is togenerate information on the probable success of treatinga given population with a selected antibiotic. Another useof the invention is to test for the presence of microbes ina mixture, solution or on a surface thought to be free ofthem. The purpose of cellular cytotoxicity testing may beto determine the susceptibility of the tumor cells to par-ticular chemotherapeutic drugs or test agents, or forscreening potential drug candidates, or to determinewhether a given drug candidate exhibits undesirable cy-totoxicity to normal cells or any indicator cell type.[0066] The method described herein is inherentlyquantitative in that the amount of the reduced form of thedye produced is proportional to the number of cells lysed.The method, however, can be used both for quantitativetesting and qualitative testing. The term qualitative test-ing refers to testing apparatus and methods which pro-duce test results that generally indicate whether an or-ganism or cellular specimen is sensitive or resistant to aparticular antibiotic or cytotoxic test agent. The relativedegree of sensitivity or resistance is not reported in qual-itative testing. The term quantitative testing refers to test-ing apparatus and methods which produce test resultsthat provide data on the concentration of the antimicrobialor cytotoxic product that will be sufficient to inhibit growthof the microorganism or other cell type. Typically, for mi-croorganism specimens, six or more different dilutionsof the test agent are utilized, covering the therapeuticrange of concentrations of the test agent. The term min-imum inhibitory concentration (MIC) is often used to referto the result provided by quantitative testing of a testagent and is defined as the minimum concentration ofthe test agent that will produce inhibition of the growth ofthe cells used in the method.[0067] The general protocol of the method proceeds

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as follows:

First, a lyophilized substrate mixture is reconstitutedin a buffer and allowed to equilibrate to assay tem-perature (usually room temperature). This yields areagent mixture that contains all of the required com-ponents to perform the method.

[0068] The reagent mixture is then added to the sam-ple(s) to be measured, the samples having been previ-ously incubated for a pre-determined time in the pres-ence of known concentrations of the test agent. The sam-ples are then gently mixed to ensure uniformity.The samples are then incubated for a predeterminedamount of time, preferably about ten minutes, prior togathering the fluorescent or colorimetric data.[0069] The reactions are stopped by the addition of asuitable stop reagent, preferably 3% w/v SDS in wateror an aqueous solution of sodium hydroxide (1 N).[0070] The fluorescence of the reduced form of theindicator dye, preferably resorufin, is then recorded. Theamount of fluorescence is indicative of the quantity ofcytoplasmic component (e.g., LDH) activity present inthe sample. The preferred filter set for fluorescent record-ing measures excitation at 560 nm and emission at 590nm. Other slight variations from these wavelengths arepossible.[0071] If resazurin is used as the dye and fluorescenceis the endpoint measured, the vessel in which the reac-tion is performed, such as microtiter assay plate, shouldbe compatible with a micro-well fluorimeter. If MTS isused as the indicator, the vessel in which the reaction isperformed should be compatible with 490 nm absorb-ance measurements.[0072] The present invention is also drawn to kits thatcontain reagents and instructions necessary to carry outthe above methods. In its most preferred form, the kitincludes an assay buffer containing a dye (again, the dyehaving an oxidized state and a reduced state whereinthe reduced state can be distinguished from the oxidizedstate and wherein the dye is present in the mixture inprimarily the oxidized state) disposed in a first container.The kit also includes a lyophilized substrate mix contain-ing an electron transfer agent, a substrate for a cytoplas-mic component (e.g., lactate) and an appropriate cofac-tor for the cytoplasmic enzyme (e.g., NAD+ in the caseof LDH) disposed in a second container. An optional thirdcontainer may hold a stop solution. An optional additionalcontainer may hold a lysis solution. The kit should alsoinclude instructions for how to use the kit.[0073] It is preferred that the substrate mixture bepresent in the form of a lyophilized powder that is thenreconstituted with a buffer to yield a reagent mix that isnon-toxic to cells and which contains the componentsrequired to practice the method of the present invention.This embodiment is preferred because the lyophilizedsubstrate mix is easier to store and transport.The kit, however, may also be configured so that the

necessary components listed above are packaged in theform of a pre-made reagent mix, ready for use.[0074] In the preferred embodiment of the kit, the dyeis resazurin or 3-(4,5-dimethylthiazol-2-yl)-5-(3-car-boxymethoxyphenyl)- 2-(4- sulphophenyl)- 2H- tetrazo-lium, inner salt (MTS). It is also preferred that the sub-strate for the cytoplasmic enzyme is lactate. It is preferredthat the electron transfer agent be an enzyme, most pref-erably diaphorase. If LDH is the cytoplasmic enzymemeasured, it is also preferred that the reagent mixturefurther contain NAD+ as the cofactor[0075] Thus, when the substrate mixture is reconsti-tuted into a reagent solution, it is preferred that the rea-gent solution comprises an aqueous solvent, lactate(e.g., lithium lactate), NAD+, diaphorase, Tris buffer,HEPES, NaCl and resazurin or MTS. More specifically,when the substrate mixture is reconstituted, it is preferredthat the reagent solution contain the following compo-nents: from about 50 mM to about 250 mM lithium lactate;from about 0.1 U/ml to about 10 U/ml diaphorase; fromabout 0.5 mM to about 10 mM NAD+; from about 1 mMto about 10 mM Tris buffer; from about 10 mM to about100 mM HEPES; from about 1 mM to about 100 mMNaCl; and from about 50 mM to about 500 mM resazurin.These components are present in a manner that is con-sistent with the required osmolality of the cells under in-vestigation (about 310 mOsm for most mammalian celltypes). Optimization of kinetics may be necessary, andare well within the skills of the typical artisan.[0076] In the most preferred embodiment, the substratemixture, when reconstituted, yields a reagent solutioncomprising the following components: about 109 mM lith-ium lactate; about 3.3 mM NAD+; about 2.2 U/ml diapho-rase; about 3.0 mM Tris buffer; about 30 mM HEPES;about 10 mM NaCl; and about 350 mM resazurin.[0077] It is preferred that when the substrate mixtureis reconstituted (or if the components are presented inthe form of a ready-made solution), that the solution havea pH offrom about 7.0 to about 8.0, more preferably fromabout 7.25 to about 7.60.[0078] The stop solution of the kit preferably is anaqueous solution of a soap, detergent, or a strong basesuch as sodium hydroxide. The preferred stop reagentis a 1% to 5% solution of SDS or sodium hydroxide (pref-erably 1N). A 3% solution of SDS is preferred.

EXAMPLES

[0079] The following Examples are provided for illus-trative purposes only. The Examples are included solelyto aid in a more complete understanding of the inventiondescribed and claimed herein. The Examples do not limitthe scope of the invention in any fashion.

Example 1: Demonstrating Background and Linearity:

[0080] A reagent solution was prepared combining alyophilized substrate mixture with the assay buffer con-

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taining resazurin, and all of the ingredients required tomeasure the release of LDH from dead cells. The buffersolvent used to reconstitute the lyophilized substrate mix-ture was designed so that when it is used to reconstitutethe mixture, the resulting solution has an osmolarity thatis compatible with living cells. That is, the resulting solu-tion was isotonic with the cells. When used to reconstitutethe lyophilized substrate mix, the reagent solution con-tained:

[0081] L929 cells were cultured in F-12/DME with 10%FBS and serially-diluted in a microtiter plate to yield du-plicate wells containing approximately 0, 1562, 3125,6250, 12,500, 25,000, and 50,000 cells per well, in 90 mltotal volume. Cells were incubated at 37˚C for sufficienttime to allow attachment (about 1.5 hours). Microtiterplactes containing cells were then removed from the in-cubator and in one set of wells, 10 ml of Triton X-100 (alysing agent) was added and lysis was allowed to pro-ceed to completion (about 30 minutes), while the cellsequilibrated to room temperature. The other set of wellswas left unlysed; 10 ml of phosphate buffered saline(PBS) was added to these as a volume control. Assayreagent mixture was prepared as previously described,and allowed to equilibrate to room temperature. Afterlysis was complete, 100ml of reagent mixture was addedto each well.The two sets of wells were allowed to incubate for 10minutes. After 10 minutes, 50 ml of a stop solution (3%SDS) was added to each well. The fluorescence of eachwell was then measured (excitation 560 nm, emission590 nm). The results are shown in Fig. 2. As can be seenfrom Fig. 2, in the wells where no lysing agent waspresent, a minimal background amount of fluorescencewas detected. In the wells where the lysing agent wasadded, the fluorescence rises in a linear fashion, propor-tional to the number of cells in each well. As can be seenfrom Fig. 2, the method yields linear results from 0 to atleast 500 cells per well to at least 50,000 cells per well.Other cell lines have been tested and show similar re-sults.[0082] This Example shows that the subject methodcan be used to calculate the cytotoxicity of a test agentbecause the amount of fluorescence detected is knownto be proportional to the number of cells lysed.

lactate: 109 mMNAD+: 3.34 mMdiaphorase: 2.17 Units/mlTris buffer: 3.05 mMHEPES: 30 mMNaCl: 10 mMresazurin: 350 mM

Example2:StabilityofLDHinCultureMediumComparedto Stability of G6PDH

[0083] Figs. 3 and 4 depict the activity of glucose-6-phosphate dehydrogenase (G6PDH) (Fig. 3) and lactatedehydrogenase (LDH) (Fig. 4) in culture medium underidentical assay conditions for two different cell types,L929 cells and Jurkat cells. The assay conditions usedwere similar to those listed above in Example 1. Formeasurement of G6PDH, glucose-6-phosphate andNADP+ were substituted for lactate and NAD+ and theconcentration of resazurin was reduced to 250 mM tohelp increase the rate of resorufin development.Previous work with this G6PDH reagent indicates thatfluorescence is proportional to the number of cells lysed.The L929 cells and Jurkat cells samples to be testedwere assembled, and a lysing agent was added. To mon-itor stability ofthe released cytoplasmic enzymes, cellswere maintained at 37˚C following addition of the lysisdetergent. The amount of fluorescence was then meas-ured at 1, 2, 4, and 6 hours from the time the lysing agentwas added. Figs. 3 and 4 thus depict the stability of LDHin culture medium as compared to the stability of G6PDHunder the same conditions. As can be seen from Fig. 4,even 6 hours after adding the lysing agent, the LDH re-leased from both L929 cells and Jurkat cells retains wellover 50% of its starting activity. In sharp contrast, as canbe seen from Fig. 3, G6PDH, under the identical condi-tions, loses more than 50% of its starting activity within2 hours of adding the lysing agent.[0084] This Example shows that in the preferred em-bodiment of the present invention, the method relies uponan enzyme, LDH, whose half-life in culture medium isgreater than 8 hours. Thus, the present invention can beused to measure cytotoxicity over longer incubation pe-riods than assays known prior to the current disclosure.

Example 3: Measuring the Cytotoxicity of TNFα Usingthe Present Invention

[0085] L929 cells (2000 cells/well) were prepared us-ing a 384-well tissue culture plate in F-12/DME mediumsupplemented with 10% horse serum. The cells wereallowed to attach and grow for 24 hours at 37˚C, 5% CO2.Various concentrations of TNFα (n = 4) in the presenceof actinomycin D (1 mg/ml final) were added to the wellsand the plate was then incubated for 20 hours. An equalvolume of reagent (25 ml/well) was then added and theplate was shaken for 30 seconds. The plate was incu-bated for 10 minutes at 22˚C, after which 12.5 ml/well of3% SDS solution was added to stop the reaction. Fluo-rescence was then determined at 560 nm excitation and590 nm emission.[0086] The results of this Example are depicted in Fig.5. As shown in Fig. 5, the amount of observed fluores-cence rose in a TNFα dose-dependent fashion at TNFconcentrations ranging from zero to about 0.125 ng/mlTNF. Above about 0.125 ng/ml, the amount of observed

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fluorescence remained essentially constant, indicatingthat all of the cells in the wells had been lysed at or abovethis concentration of TNFα. These results indicate thatthe invention measures fluorescence following cell lysisat a rate proportional to lysis by a known cytotoxic agent.

Claims

1. A method for determining cytotoxicity of a test agent,the method comprising:

(a) contacting living cells in culture medium con-tained in a vessel, with a predetermined amountof the test agent; and(b) incubating the cells in culture medium fromStep (a) for a pre-determined amount of time;then(c) adding to the cells and culture medium fromStep (b) a reagent mixture that is non-toxic tothe living cells, wherein the reagent mixturecomprises:

a solvent, a dye having an oxidized stateand a reduced state wherein the reducedstate can be distinguished from the oxidizedstate and wherein the dye is initially presentin the oxidized state, an electron transferagent, a substrate for a cytoplasmic enzymehaving a half-life greater than two hours,and a cofactor for the cytoplasmic enzyme;and then

(d) measuring the culture medium from Step (c)for production of the reduced state of the dye,the production of the reduced state of the dyebeing caused by a cytoplasmic enzyme specificfor the substrate in the reagent mixture addedin Step (c), wherein the cytoplasmic enzyme be-ing released from dead and dying cells withinthe vessel, whereby the cytotoxicity of the testagent is determined.

2. A method as claimed in claim 1, wherein in Step (c),the reduced state of the dye can be distinguishedfrom the oxidized state of the dye fluorimetrically,and in Step (d), the culture medium is measured forthe production of the reduced state of the dye viafluorescent spectroscopy.

3. A method as claimed in claim 1, wherein in Step (c),the reduced state of the dye can be distinguishedfrom the oxidized state of the dye colorimetrically,and in Step (d), the culture medium is measured forthe production of the reduced state of the dye viavisible spectroscopy.

4. A method as clamed in claim 1, wherein in Step (c),

the dye is resazurin or 3-(4,5-dimethylthiazol-2-yl)-5-(3- carboxymethoxyphenyl)- 2-(4- sulphophenyl)-2H-tetrazolium inner salt (MTS).

5. A method as claimed in claim 1, wherein in Step (c),the electron transfer agent is an enzyme.

6. A method as claimed in claim 1, wherein in Step (c),the electron transfer agent is diaphorase.

7. A method as claimed in claim 1, wherein in Step (c),the substrate is lactate and the cofactor is NAD+,and in Step (d), the production of the reduced stateof the dye is caused by lactate dehydrogenase.

8. A method as claimed in claim 1, wherein in Step (a),the vessel contains living cells that are prokaryotic.

9. A method as claimed in claim 1, wherein in Step (a),the vessel contains living cells that are eukaryotic.

10. A method as claimed in claim 1, wherein in Step (a),the vessel contains living cells that are cultured eu-karyotic cells.

11. A method as claimed in claim 1, wherein in Step (a),the test agent is a known antibiotic.

12. A method as claimed in claim 1, wherein in Step (a),the test agent is a known pharmaceutical or thera-peutic agent.

13. A method as claimed in claim 1, wherein: in Step (b)the cells are incubated, in Step (c) the reagent mix-ture is added, and in Step (d), the culture medium ismeasured for production of the reduced state of thedye, all in the vessel from Step (a).

14. A method as claimed in claim 1, wherein in Step (d),the culture medium is measured for production ofthe reduced state of the dye without separating theculture medium from the cells from Step (c).

15. A method as claimed in claim 1, wherein in Step (c),the solvent is an aqueous solvent.

16. A method as claimed in claim 15, wherein in Step(c), the solvent is a buffered solvent.

17. A method as claimed in claim 1, wherein the reagentmixture comprises an aqueous solvent, lactate,NAD+, diaphorase, Tris buffer, HEPES, NaCl andresazurin.

18. A method as claimed in claim 17, wherein the rea-gent mixture comprises:

from 50 mM to 250 mM lactate;

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from 0.1U/ml to 10U/ml diaphorase;from 0.5 mM to 10 mM NAD+;from 1 mM to 10 mM Tris buffer;from 10 mM to 100 mM HEPES;from 1 mM to 100 mM NaCl; andfrom 50 mM to 500 mM resazurin.

19. A method as claimed in claim 17, wherein the rea-gent mixture comprises

109 mM lactate;3. 3 mM NAD+;2.2U/ml diaphorase3.0 mM Tris buffer;30 mM HEPES;10 mM NaCl; and350mM resazurin.

20. A method as claimed in claim 1, wherein the reagentmixture has a pH of from 7.0 to 8.0.

21. A method as claimed in claim 1, wherein the reagentmixture has a pH of from 7.25 to 7.60.

22. A method as claimed in claim 1, further comprisingthe step of, after Step (c) and prior to Step (d), addinga stop solution to the vessel.

23. A method as claimed in claim 22, wherein the stopsolution comprises a soap or a detergent.

24. A method as claimed in claim 22, wherein the stopsolution comprises sodium dodecylsulphate.

25. A method as claimed in claim 22, wherein the stopsolution comprises sodium hydroxide.

26. A method as claimed in claim 1, wherein in Step (c)the reagent mixture comprises a substrate for a cy-toplasmic enzyme that has a half-life greater thantwo hours following cell lysis or cell death at 37˚C.

27. A method as claimed in claim 1, wherein in Step (c)the reagent mixture comprises a substrate for a cy-toplasmic enzyme that has a half-life greater thanfour hours following cell lysis or cell death at 37˚C.

28. A method as claimed in claim 1, wherein in Step (c)the reagent mixture comprises a substrate for a cy-toplasmic enzyme that has a half-life greater thansix hours following cell lysis or cell death at 37˚C.

29. A method as claimed in claim 1, wherein in Step (c)the reagent mixture comprises a substrate for a cy-toplasmic enzyme that has a half-life greater thaneight hours following cell lysis or cell death at 37˚C.

30. A method as claimed in any preceding claim wherein

in Step (a) a pre-determined amount of the test agentis added to a vessel containing living cells in culturemedium; and the reagent mixture of Step (c) com-prises:an aqueous solvent, resazurin, an electron transfer-ring agent, NAD+ and lactate; and Step (d) compris-es measuring the culture medium from Step (c) forproduction of resorufin without removing the mediumfrom the vessel, the production of resorufin beingcaused by lactate dehydrogenase released fromdead and dying cells within the vessel, whereby thecytotoxicity of the test agent is determined.

31. A method as claimed in claim 30, wherein in Step(c), the electron transfer agent is Meldola’s Blue.

32. A method as claimed in claim 30, wherein in Step(a), the test agent is a known or putative toxic com-pound.

33. A method as claimed in claim 30, wherein in Step(d), the production of resorufin is measured fluori-metrically.

34. A method as claimed in claim 30, wherein in Step(d), the production of resorufin is measured colori-metrically.

35. A method as claimed in claim 30, wherein the rea-gent mixture comprises an aqueous solvent, lactate,NAD+, diaphorase, Tris buffer, HEPES, NaCl andresazurin.

36. A kit for measuring cytotoxicity of a test agent, thekit comprising, in combination:

a dye disposed in a first container, the dye hav-ing an oxidized state and a reduced state

wherein the reduced state can be distinguished fromthe oxidized state and wherein the dye is present inthe oxidized state, said dye being from 50mM to500mM resazurin,wherein the resazurin is disposed in a premixed,ready-to-use reagent mixture that is non toxic to liv-ing cells, the reagent mixture comprising from0.1U/ml to 10 U/ml diaphorase, from 50mM to250mM lactate , from 0.5 mM to 10mM NAD+, from1mM to 10mM Tris buffer, from 10mM to 100mMHEPES, and from 1mM to 100mM NaCl; said sub-strate mix having a pH of from 7.0 to 8.0 in solution,and instructions for use of the kit.

37. A kit as claimed in claim 36, wherein the reducedstate of the dye can be distinguished from the oxi-dized state of the dye fluorimetrically or colorimetri-cally.

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38. A kit as claimed in claim 36, wherein the reagentmixture comprises an aqueous solution comprising:

109 mM lactate;3.3 mM NAD+;2.2 U/ml diaphorase3.0 mM Tris buffer;30 mM HEPES;10 mM NaCl; and350 mM resazurin.

39. A kit as claimed in claim 36, wherein the reagentmixture has a pH of from 7.25 to 7.60.

40. A kit as claimed in claim 36, further comprising astop solution disposed in a second container.

41. A kit as claimed in claim 40, wherein the stop solutioncomprises a soap or a detergent.

42. A kit as claimed in claim 40, wherein the stop solutioncomprises sodium dodecylsulphate.

43. A kit as claimed in claim 40, wherein the stop solutioncomprises sodium hydroxide.

Patentansprüche

1. Verfahren zur Bestimmung der Zytotoxizität einesTestagens, wobei das Verfahren umfasst:

(a) in Kontakt bringen von lebenden Zellen ineinem Kulturmedium in einem Gefäß mit einervorbestimmten Menge des Testagens; und(b) Inkubieren der Zellen in dem Kulturmediumgemäß Schritt (a) über eine vorbestimmte Zeit-dauer; dann(c) Zusetzen einer Reagenzmischung zu denZellen und dem Kulturmedium gemäß Schritt(b); die für die lebenden Zellen nicht toxisch ist,wobei die Reagenzmischung enthält:

ein Lösungsmittel, einen Farbstoff mit ei-nem oxidierten Zustand undeinem reduzierten Zustand, wobei der re-duzierte Zustand von dem oxidierten Zu-stand unterschieden werden kann, und

wobei der Farbstoff zu Beginn im oxidierten Zu-stand vorliegt, ein Elektronentransfermittel, einSubstrat für ein zytoplasmatisches Enzym, daseine Halbwertszeit von mehr als zwei Stundenhat, und einen Co-Faktor für das zytoplasmati-sche Enzym; und dann(d) Vermessen des Kulturmediums gemäßSchritt (c) auf die Ausbildung des reduziertenZustandes des Farbstoffes, wobei die Ausbil-

dung des reduzierten Zustandes des Farbstof-fes durch ein zytoplasmatisches Enzym verur-sacht ist, das für das Substrat in der Reagenz-mischung spezifisch ist, die in Schritt (c) zuge-setzt wird,

wobei das zytoplasmatische Enzym von toten odersterbenden Zellen in dem Gefäß freigesetzt wird,wodurch die Zytotoxizität des Testagens bestimmtwird.

2. Verfahren nach Anspruch 1,wobei in Schritt (c) der reduzierte Zustand des Farb-stoffes von dem oxidierten Zustand des Farbstoffesfluorimetrisch unterschieden werden kann, und inSchritt (d), die Vermessung des Kulturmediums aufdie Ausbildung des reduzierten Zustandes des Farb-stoffes mittels Fluoreszenzspektroskopie erfolgt.

3. Verfahren nach Anspruch 1,wobei in Schritt (c) der reduzierte Zustand des Farb-stoffes von dem oxidierten Zustand des Farbstoffeskolorimetrisch unterschieden werden kann, und inSchritt (d), die Vermessung des Kulturmediums aufdie Ausbildung des reduzierten Zustandes des Farb-stoffes mittels sichtbarer Spektroskopie erfolgt.

4. Verfahren nach Anspruch 1,wobei in Schritt (c) der Farbstoff Resazurin oder in-neres 3-(4,5-Dimethylthiazol-2-yl)-5-(3-carboxyme-thoxyphenyl)- 2-(4- sulphophenyl)- 2H- tetrazolium-salz (MTS) ist.

5. Verfahren nach Anspruch 1,wobei in Schritt (c) das Elektronentransfermittel einEnzym ist.

6. Verfahren nach Anspruch 1,wobei in Schritt (c) das Elektronentransfermittel Dia-phorase ist.

7. Verfahren nach Anspruch 1,wobei in Schritt (c) das Substrat Laktat und der Co-Faktor NAD+ ist, und in Schritt (d) die Ausbildungdes reduzierten Zustandes Farbstoffes durch Lak-tatDehydrogenase bewirkt wird.

8. Verfahren nach Anspruch 1,wobei in Schritt (a) das Gefäß prokaryotische leben-de Zellen enthält.

9. Verfahren nach Anspruch 1,wobei in Schritt (a) das Gefäß eukaryotische leben-de Zellen enthält.

10. Verfahren nach Anspruch 1,wobei in Schritt (a) das Gefäß lebende Zellen enthält,die kultivierte eurkaryotische Zellen sind.

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11. Verfahren nach Anspruch 1,wobei in Schritt (a) das Testagens ein bekanntesAntibiotikum ist.

12. Verfahren nach Anspruch 1,wobei in Schritt (a) das Testagens ein bekanntespharmazeutisches oder therapeutisches Mittel ist.

13. Verfahren nach Anspruch 1,wobei jeweils in dem Gefäß gemäß Schritt (a) inSchritt (b) die Zellen inkubiert werden, in Schritt (c)die Reagenzmischung zugesetzt wird, und in Schritt(d) das Kulturmedium auf die Ausbildung des redu-zierten Zustandes des Farbstoffes hin vermessenwird.

14. Verfahren nach Anspruch 1,wobei in Schritt (d) das Vermessen des Kulturmedi-ums auf die Ausbildung des reduzierten Zustandesdes Farbstoffes hin ohne Abtrennung des Kulturme-diums von den Zellen gemäß Schritt (c) erfolgt.

15. Verfahren nach Anspruch 1,wobei in Schritt (c) das Lösungsmittel ein wässrigesLösungsmittel ist.

16. Verfahren nach Anspruch 15,wobei in Schritt (c) das Lösungsmittel ein gepuffertesLösungsmittel ist.

17. Verfahren nach Anspruch 1,wobei die Reagenzmischung ein wässriges Lö-sungsmittel, Laktat, NAD+, Diaphorase, Tris-Puffer,HEPES, NaCl und Resazurin enthält.

18. Verfahren nach Anspruch 17,wobei die Reagenzmischung enthält:

50 mM bis 250 mM Laktat;0,1 U/ml bis 10 U/ml Diaphorase;0,5 mM bis 10 mM NAD+;1 mM bis 10 mM Tris-Puffer;10 mM bis 100 mM HEPES;1 mM bis 100 mM NaCl; und50 mM bis 500 mM Resazurin.

19. Verfahren nach Anspruch 17,wobei die Reagenzmischung enthält:

109 mM Laktat;3,3 mM NAD+;2,2 U/ml Diaphorase;3,0 mM Tris-Puffer;30 mM HEPES;10 mM NaCl; und350 mM Resazurin.

20. Verfahren nach Anspruch 1,

wobei die Reagenzmischung einen pH-Wert von 7,0bis 8,0 hat.

21. Verfahren nach Anspruch 1,wobei die Reagenzmischung einen pH-Wert von7,25 bis 7,60 hat.

22. Verfahren nach Anspruch 1,das nach Schritt (c) und vor Schritt (d) zusätzlich denSchritt aufweist gemäß dem, dem Gefäß eine Stopp-lösung zugesetzt wird.

23. Verfahren nach Anspruch 22,wobei die Stopplösung eine Seife oder ein Deter-gens enthält.

24. Verfahren nach Anspruch 22,wobei die Stopplösung Natriumdodecylsulphat ent-hält.

25. Verfahren nach Anspruch 22,wobei die Stopplösung Natriumhydroxid enthält.

26. Verfahren nach Anspruch 1,wobei in Schritt (c) die Reagenzmischung ein Sub-strat für ein zytoplasmatisches Enzym enthält, daseine Halbwertszeit hat, die mehr als zwei Stundennach der Zelllysis oder dem Zelltod bei 37 ˚C beträgt.

27. Verfahren nach Anspruch 1,wobei in Schritt (c) die Reagenzmischung ein Sub-strat für ein zytoplasmatisches Enzym enthält, daseine Halbwertszeit hat, die mehr als vier Stundennach der Zelllysis oder dem Zelltod bei 37 ˚C beträgt.

28. Verfahren nach Anspruch 1,wobei in Schritt (c) die Reagenzmischung ein Sub-strat für ein zytoplasmatisches Enzym enthält, daseine Halbwertszeit hat, die mehr als sechs Stundennach der Zelllysis oder dem Zelltod bei 37 ˚C beträgt.

29. Verfahren nach Anspruch 1,wobei in Schritt (c) die Reagenzmischung ein Sub-strat für ein zytoplasmatisches Enzym enthält, daseine Halbwertszeit hat, die mehr als acht Stundennach der Zelllysis oder dem Zelltod bei 37 ˚C beträgt.

30. Verfahren nach einem der vorhergehenden Ansprü-che,wobei in Schritt (a) eine vorbestimmte Menge anTestagens zu einem Gefäß mit lebenden Zellen ineinem Kulturmedium gegeben wird; und wobei dieReagenzmischung gemäß Schritt (c) enthält:ein wässriges Lösungsmittel, Resazurin, ein Elek-tronentransfermittel, NAD+ und Laktat; und Schritt(d) das Vermessen des Kulturmediums gemäßSchritt (c) auf die Ausbildung von Resorufin umfasst,ohne dass das Medium aus dem Gefäß entfernt wird,

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wobei die Erzeugung von Resorufin durch Laktat-Dehydrogenase bewirkt wird, die von toten und ster-benden Zellen in dem Gefäß freigesetzt werden, so-dass die Zytotoxizität des Testagens bestimmt wird.

31. Verfahren nach Anspruch 30,wobei in Schritt (c) das Elektronentransfermittel Mel-dola’s Blue ist.

32. Verfahren nach Anspruch 30,wobei in Schritt (a) das Testagens eine bekannteoder putative toxische Verbindung ist.

33. Verfahren nach Anspruch 30,wobei in Schritt (d) die Ausbildung von Resorufinfluorimetrisch gemessen wird.

34. Verfahren nach Anspruch 30,wobei in Schritt (d) die Ausbildung von Resorufinkolorimetrisch gemessen wird.

35. Verfahren nach Anspruch 30,wobei die Reagenzmischung ein wässriges Lö-sungsmittel, Laktat, NAD+, Diaphorase, Tris-Puffer,HEPES, NaCl und Resazurin enthält.

36. Kit zum Messen der Zytotoxizität eines Testagens,wobei das Kit in Kombination enthält:

einen Farbstoff in einem ersten Behälter, wobeider Farbstoff einen oxidierten Zustand und ei-nen reduzierten Zustand hat, wobei der redu-zierte Zustand von dem oxidierten Zustand un-terschieden werden kann, und

wobei der Farbstoff in dem oxidierten Zustand vor-liegt, wobei der Farbstoff Resazurin in einer Mengevon 50 mM bis 500 mM ist, wobei das Resazurin ineiner vorgefertigten einsatzbereiten Reagenzmi-schung vorliegt, die nicht toxisch für lebende Zellenist, wobei die Reagenzmischung 0,1 U/ml bis 10 U/mlDiaphorase, 50 mM bis 250 mM Laktat, 0,5 mM bis10 mM NAD+, 1mM bis 10 mM Tris-Puffer, 10 mMbis 100 mM HEPES, und 1 mM bis 100 mM NaClenthält, wobei der Substratmix einen pH-Wert von7,8 bis 8,0 in Lösung aufweist, und eine Bedienungs-anleitung zur Benutzung des Kits.

37. Kit nach Anspruch 36,wobei der reduzierte Zustand des Farbstoffs vondem oxidierten Zustand des Farbstoffs fluorome-trisch oder kolorimetrisch unterschieden werdenkann.

38. Kit nach Anspruch 36,wobei die Reagenzmischung eine wässrige Lösungenthält, die enthält:

109 mM Laktat;3,3 mM NAD+;2,2 U/ml Diaphorase;3,0 mM Tris-Puffer;30 mM HEPES;10 mM NaCl; und350 mM Resazurin.

39. Kit nach Anspruch 36,wobei die Reagenzmischung einen pH-Wert von7,25 bis 7,60 hat.

40. Kit nach Anspruch 36,zusätzlich einen zweiten Behälter mit einer Stopplö-sung enthaltend.

41. Kit nach Anspruch 40,wobei die Stopplösung eine Seife oder ein Deter-gens enthält.

42. Kit nach Anspruch 40,wobei die Stopplösung Natriumdodecylsulphat ent-hält.

43. Kit nach Anspruch 40,wobei die Stopplösung Natriumhydroxid enthält.

Revendications

1. Méthode de détermination de la cytotoxicité d’unagent d’essai, la méthode comprenant :

(a) la mise en contact de cellules vivantes dansun milieu de culture contenu dans un récipient,avec une quantité prédéterminée de l’agentd’essai ; et(b) l’incubation des cellules dans le milieu deculture de l’étape (a) pendant une duréeprédéterminée ; puis(c) l’ajout aux cellules et au milieu de culture del’étape (b) d’un mélange réactif qui est non toxi-que pour les cellules vivantes, dans lequel lemélange réactif comprend :

un solvant, un colorant ayant un état oxydéet un état réduit dans lequel l’état réduit peutêtre distingué de l’état oxydé et dans lequelle colorant est initialement présent à l’étatoxydé, un agent de transfert d’électrons, unsubstrat pour une enzyme cytoplasmiqueayant une demi-vie supérieure à deux heu-res, et un cofacteur pour l’enzymecytoplasmique ; et ensuite

(d) la mesure du milieu de culture de l’étape (c)pour la production de l’état réduit du colorant, laproduction de l’état réduit du colorant étant pro-

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voquée par une enzyme cytoplasmique spécifi-que pour le substrat dans le mélange réactifajouté à l’étape (c), dans lequel l’enzyme cyto-plasmique est libérée des cellules mortes et vi-vantes à l’intérieur du récipient, grâce à quoi lacytotoxicité de l’agent d’essai est déterminée.

2. Méthode selon la revendication 1, dans laquelle àl’étape (c), l’état réduit du colorant peut être distinguéde l’état oxydé du colorant fluorimétriquement, et àl’étape (d), le milieu de culture est mesuré pour laproduction de l’état réduit du colorant via une spec-troscopie fluorescente.

3. Méthode selon la revendication 1, dans laquelle àl’étape (c), l’état réduit du colorant peut être distinguéde l’état oxydé du colorant colorimétriquement, et àl’étape (d), le milieu de culture est mesuré pour laproduction de l’état réduit du colorant via une spec-troscopie visible.

4. Méthode selon la revendication 1, dans laquelle àl’étape (c), le colorant est de la résazurine ou un selinterne de 3-(4,5-diméthylthiazol-2-yl)-5-(3-car-boxyméthoxyphényl)-2-(4-sulfophényl)-2H-tétrazo-lium (MTS).

5. méthode selon la revendication 1, dans laquelle àl’étape (c), l’agent de transfert d’électrons est uneenzyme.

6. Méthode selon la revendication 1, dans laquelle àl’étape (c), l’agent de transfert d’électrons est unediaphorase.

7. Méthode selon la revendication 1, dans laquelle àl’étape (c), le substrat est le lactate et le cofacteurest NAD+, et à l’étape (d), la production de l’étatréduit du colorant est provoquée par la déshydrogé-nase lactique.

8. Méthode selon la revendication 1, dans laquelle àl’étape (a), le récipient contient des cellules vivantesqui sont procaryotiques.

9. Méthode selon la revendication 1, dans laquelle àl’étape (a), le récipient contient des cellules vivantesqui sont eucaryotiques.

10. Méthode selon la revendication 1, dans laquelle àl’étape (a), le récipient contient des cellules vivantesqui sont des cellules eucaryotiques de culture.

11. Méthode selon la revendication 1, dans laquelle àl’étape (a), l’agent d’essai est un antibiotique connu.

12. Méthode selon la revendication 1, dans laquelle àl’étape (a), l’agent d’essai est un agent thérapeuti-

que ou pharmaceutique connu.

13. Méthode selon la revendication 1, dans laquelle : àl’étape (b) les cellules sont incubées, à l’étape (c) lemélange réactif est ajouté, et à l’étape (d), le milieude culture est mesuré pour la production de l’étatréduit du colorant, tous se trouvant dans le récipientde l’étape (a).

14. Méthode selon la revendication 1, dans laquelle àl’étape (d), le milieu de culture est mesuré pour laproduction de l’état réduit du colorant sans séparerle milieu de culture des cellules de l’étape (c).

15. Méthode selon la revendication 1, dans laquelle àl’étape (c), le solvant est un solvant aqueux.

16. Méthode selon la revendication 15, dans laquelle àl’étape (c), le solvant est un solvant tamponné.

17. Méthode selon la revendication 1, dans laquelle lemélange réactif comprend un solvant aqueux, lac-tate, NAD+, diaphorase, un tampon Tris, HEPES,NaCl et résazurine.

18. Méthode selon la revendication 17, dans laquelle lemélange réactif comprend :

entre 50 mM et 250 mM de lactate ;entre 0,1 U/ml et 10 U/ml de diaphorase ;entre 0,5 mM et 10 mM de NAD+ ;entre 1 mM et 10 mM de tampon Tris ;de 10 mM à 100 mM d’HEPES ;de 1 mM à 100 mM de NaCl ; etde 50 mM à 500 mM de résazurine.

19. Méthode selon la revendication 17, dans laquelle lemélange réactif comprend

109 mM de lactate ;3,3 mM de NAD+ ;2,2 U/ml de diaphorase ;3,0 mM de tampon Tris ;30 mM d’HEPES ;10 mM de NaCl ; et350 mM de résazurine.

20. Méthode selon la revendication 1, dans laquelle lemélange réactif a un pH compris entre 7,0 et 8,0.

21. Méthode selon la revendication 1, dans laquelle lemélange réactif a un pH compris entre 7,25 et 7,60.

22. Méthode selon la revendication 1, comprenant enoutre l’étape consistant à, après l’étape (c) et avantl’étape (d), ajouter une solution d’arrêt dans le réci-pient.

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23. Méthode selon la revendication 22, dans laquelle lasolution d’arrêt comprend un savon ou un détergent.

24. Méthode selon la revendication 22, dans laquelle lasolution d’arrêt comprend du dodécylsulfate de so-dium.

25. Méthode selon la revendication 22, dans laquelle lasolution d’arrêt comprend de l’hydroxyde de sodium.

26. Méthode selon la revendication 1, dans laquelle àl’étape (c), le mélange réactif comprend un substratpour une enzyme cytoplasmique qui a une demi-viesupérieure à deux heures à la suite de la lyse descellules ou de la mort des cellules à 37˚C.

27. Méthode selon la revendication 1, dans laquelle àl’étape (c), le mélange réactif comprend un substratpour une enzyme cytoplasmique qui a une demi-viesupérieure à quatre heures à la suite de la lyse descellules ou de la mort des cellules à 37˚C.

28. Méthode selon la revendication 1, dans laquelle, àl’étape (c), le mélange réactif comprend un substratpour une enzyme cytoplasmique qui a une demi-viesupérieure à six heures à la suite de la lyse des cel-lules ou de la mort des cellules à 37˚C.

29. Méthode selon la revendication 1, dans laquelle, àl’étape (c), le mélange réactif comprend un substratpour une enzyme cytoplasmique qui a une demi-viesupérieure à huit heures à la suite de la lyse descellules ou de la mort des cellules à 37˚C.

30. Méthode selon l’une quelconque des revendicationsprécédentes dans laquelle, à l’étape (a), une quan-tité prédéterminée de l’agent d’essai est ajoutéedans un récipient contenant des cellules vivantesdans un milieu de culture ; et le mélange réactif del’étape (c) comprend :un solvant aqueux, résazurine, un agent de transfertd’électrons, NAD+ et lactate ; et l’étape (d) com-prend la mesure du milieu de culture de l’étape (c)pour la production de résorufine sans retirer le milieudu récipient, la production de résorufine étant pro-voquée par la déshydrogénase lactique dégagéedes cellules mortes et mourantes à l’intérieur du ré-cipient, grâce à quoi la cytotoxicité de l’agent d’essaiest déterminée.

31. Méthode selon la revendication 30, dans laquelle, àl’étape (c), l’agent de transfert d’électrons est du bleude Meldola.

32. Méthode selon la revendication 30, dans laquelle àl’étape (a), l’agent d’essai est un composé toxiqueconnu ou putatif.

33. Méthode selon la revendication 30, dans laquelle àl’étape (d), la production de résorufine est mesuréefluorimétriquement.

34. Méthode selon la revendication 30, dans laquelle àl’étape (d), la production de résorufine est mesuréecolorimétriquement.

35. Méthode selon la revendication 30, dans laquelle lemélange réactif comprend un solvant aqueux, lac-tate, NAD+, diaphorase, un tampon Tris, HEPES,NaCl et résazurine.

36. Kit de mesure de la cytotoxicité d’un agent d’essai,le kit comprenant, en combinaison :un colorant disposé dans un premier conteneur, lecolorant ayant un état oxydé et un état réduit, danslaquelle l’état réduit peut être distingué de l’état oxy-dé et dans laquelle le colorant est présent à l’étatoxydé, ledit colorant étant de 50 mM à 500 mM derésazurine, dans laquelle la résazurine est disposéedans un mélange réactif prêt à l’emploi qui n’est pastoxique pour les cellules vivantes, le mélange réactifcomprenant entre 0,1 U/ml et 10 U/ml de diaphorase,entre 50 mM et 250 mM de lactate, entre 0,5 mM et10 mM de NAD+, entre 1 mM et 10 mM de tamponTris, entre 10 mM et 100 mM de HEPES et entre 1mM et 100 mM de NaCl ; ledit mélange de substratayant un pH compris entre 7,0 et 8,0 en solution, etdes instructions pour l’utilisation du kit.

37. Kit selon la revendication 36, dans lequel l’état réduitdu colorant peut être distingué de l’état oxydé ducolorant fluorimétriquement ou colorimétriquement.

38. Kit selon la revendication 36, dans lequel le mélangeréactif comprend une solution aqueusecomprenant :

109 mM de lactate ;3,3 mM de NAD+ ;2,2 U/ml de diaphorase ;3,0 mM de tampon Tris ;30 mM d’HEPES ;10 mM de NaCl ; et350 mM de résazurine.

39. Kit selon la revendication 36, dans lequel le mélangeréactif a un pH compris entre 7,25 et 7,60.

40. Kit selon la revendication 36, comprenant en outreune solution d’arrêt disposée dans un second con-teneur.

41. Kit selon la revendication 40, dans lequel la solutiond’arrêt comprend un savon ou un détergent.

42. Kit selon la revendication 40, dans lequel la solution

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d’arrêt comprend du dodécylsulfate de sodium.

43. Kit selon la revendication 40, dans lequel la solutiond’arrêt comprend de l’hydroxyde de sodium.

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REFERENCES CITED IN THE DESCRIPTION

This list of references cited by the applicant is for the reader’s convenience only. It does not form part of the Europeanpatent document. Even though great care has been taken in compiling the references, errors or omissions cannot beexcluded and the EPO disclaims all liability in this regard.

Patent documents cited in the description

• US 5501959 A, Lancaster [0008] [0017]• US 5185450 A [0012]

• CA 1112140 [0018]• US 3107204 A [0018]

Non-patent literature cited in the description

• Corey et al. J. Immunol. Meth., 1997, vol. 207, 43-51[0004]

• Buttke et al. J. Immunol Methods, 1993, vol. 157,233-240 [0006]

• Dunigan et al. BioTechniques, 1995, vol. 19,640-649 [0006]

• Baker et al. Microbiol., 1980, vol. 26, 248-253 [0018]• Kanazawa et al. J. Antibiotics, 1966, vol. 19, 229-233

[0018]• Brown et al. J. Clin. Path., 1961, vol. 5, 10-13 [0018]