LDH Cytotoxicity Detection Kit User Manual
Contents
1. Test substance or assay medium have LDH activity Use Substance Control to measure test substance and or assay medium for intrinsic LDH activity Spontaneous LDH release caused by cell death due to assay conditions Check culture conditions some cell lines do not sur vive in serum free media even for short incubation times Increase serum concentration to about 1 5 Strong color reaction but low absorbance values Test substance or assay medium have LDH activity Use Substance Control to measure test substance and or assay medium for intrinsic LDH activity Strong color reaction in Effector Cell Controls Spontaneous LDH release caused by effector cell damage or death due to culture condi tions or isolation method Improve cell culture conditions Separate viable from dead effector cells by density gradient centrifugation e Substance Control I Measures the LDH activity contained in the test substance or effector cells The proce dure to perform Substance Control I is described in detail in the Part VI Protocols e Substance Control II Determines whether the test substance itself interferes with LDH activity To perform this control proceed as follows a Add 50 pl test substance solution diluted in assay medium 50 pl LDH solution 0 05 U ml and 100 ul freshly prepared Reaction Mixture Part V A to triplicate wells Add 50 pl assay medium 50 pl LDH solution 0 05 U ml and 12. Measure absorbance at 490 or 492 nm reference wavelength greater than 600 nm All assays must be performed in triplicate A Protocol Preparing Cell Suspensions to Measure Cytotoxicity Due to Soluble Compounds 1 Titrate test substance in triplicate in assay medium into wells B1 G6 of a sterile 96 well plate by twofold serial dilution final volume 100 pl well Note Do not add test substance to the Control wells A1 A6 and H1 H6 see Table IV Wash the cells in assay medium and dilute the cell suspension to the optimal concentration determined in Part V B Add 100 pl well cell suspension to the dilutions of the test substance Note Do not add cells to the Control wells A1 A6 and H1 H6 see Table IV Prepare the following controls on the plate See Table IV a Background Control Add 200 pl assay medium to triplicate wells Al A3 b Substance Control I Add 100 pl test substance at the maximum concentration used in the experiment and 100 ul assay medium to triplicate wells A4 A6 c Low Control for spontaneous LDH release Add 100 pl cell suspension and 100 pl assay medium to triplicate wells H1 H3 d High Control for maximum LDH release Add 100 pl cell suspension and 100 pl Triton X 100 solution to triplicate wells H4 H6 Proceed to Protocol E Protocol No PT3947 1 Version No 9 PR6Y2138 www clontech com Clontech Laboratories Inc ATakara Bio Company LDH Cytotoxicity Dete
3. User Manual LDH Cytotoxicity Detection Kit User Manual O Clontech United States Canada 800 662 2566 Asia Pacific 1 650 919 7300 Europe 33 0 1 3904 6880 Japan 81 0 77 543 6116 Cat No 630117 Clontech Laboratories Inc ATakara Bio Company PT3947 1 PR6Y2138 1290 Terra Bella Ave Mountain Vion CA 94045 Published 17 January 2007 Technical Support US E mail tech clontech com www clontech com LDH Cytotoxicity Detection Kit Table of Contents le IMtrOGUCTION 0 00 cee eeteeeeeeeeeeeeeeeeeeeeeeeeeeeeseeeeseeeeseeesneeescessseessoessegeseeeeseeeesaeeseneesneeeseessseensesasenees 2 Is List Of Components is siceccscissceceessssceccecd iscicestssdscodsscessseedensssesenedadsabocstdavscessexssaandecddadsabensdtsiacens 4 Ill Additional Materials Required ccccccececceeeeeceee eee eeee eee eeeeeeeaeeaeeeeeeeeeeeeeeeeessaaeeaeeeseeeeeeeeeees 5 IV LDH Cytotoxicity Detection Kit Protocol Overview cccccscsceecsseeeeeeeeseeeeeeessseeeeeeeseneees 6 A Protocol VEL VIC Wero a ace ccu teh ease castes tae veka gp Me ACORN E CE Y 6 B Experiment Controls ersero e e Ea RET T EEE IREE N EDORAS ESAE ESN 6 V Sample Preparation and Experiment Setup c ccccccssssseeeeeseeeeeeeseeeeeeeessaeeeeeeeeaeeeeeeee 7 A Protocol Preparing Working Solutionsi nenene E cadence eco vente 7 B Protocol Determining the Optimal Cell Concentration 0 ceesecsseseeescneeeeeeeeseeenseasseeeneeeseeneesesanaes 7
4. VI Measuring Cytotoxicity iiiiincccccccsesscnascencsccenccecscensssteaeesnoxseeseceasschessnsancenectesscees lt cesdancencnaneieneseesss 9 A Protocol Preparing Cell Suspensions to Measure Cytotoxicity Due to Soluble Compounds 9 B Protocol Preparing Adherent Cells to Measure Cytotoxicity Due to Soluble Compounds 45 10 C Protocol Preparing Samples to Measure Cell Mediated Cytotoxicity ceccccesesensesesesenseeeeeeneeesee 11 D Protocol Assaying Cell Death in Fermentation Cultures ccecscsseseeeecseseeeseeseeseeesesenseeeesseneeeeees 11 E Protocol Measuring LOH Releases scisc cisestissiahhvarpsrcotestnits E EE E 12 E Protocol Calculating Cytotoxicity Due to Soluble Substances wc eceeseeseseeeeeesesenseeeseeenseeeeeneneesee 12 G Protocol Calculating Percent Cell Mediated Cytotoxicity cccecsesseeeecreteeeteeseteseeeseeeseeeesseneeetees 12 VIL TroubleSNOOTING wescsiscctccnessc cectevevescacceesccccuserevesccvensenecaucunniseccecenivarvennianvesstaenenedsceeresvacencenneeeccts 13 VIN Referents oi ie vecsatstia Fetesdacanctinedescessa cubis ciewntetanadsanags tees onuea ciseatssbansteanacvucsujnninacsiesactbansinaniasays 14 Appendix A Examples of LDH Cytotoxicity Detection Kit Results cccccccsssseeeeesseeees 15 A Optimuzitio Cell Concentra tons gsesnscsescstzicsessesstescctivess rA e N Ee TEREE ESETE e ESER ENE 15 B Measuring Detergent Cytotoxicity oisises setcesestscucdsnebesctensstcnsecesedoes esodn
5. cell medi ated toxicity is measured this control provides information on the LDH activity released from the effector cells e g NK cells LAK cells CTLs e Substance Control II Determines whether the test substance itself interferes with LDH activity Note Perform all assays including controls in triplicate Clontech Laboratories Inc www clontech com Protocol No PT3947 1 ATakara Bio Company Version No PR6Y2138 6 LDH Cytotoxicity Detection Kit V Sample Preparation and Experiment Setup A Protocol Preparing Working Solutions Prepare working solutions as directed in Table II Table II Working Solutions Solution Preparation Storage and Stability Catalyst bottle 1 Reconstitute the lyophilate in 1 ml distilled water Several weeks at 4 C blue cap and mix thoroughly for 10 min Dye Solution bottle 2 Thawed INT dye solution is ready to use Several weeks at 4 C Recipes red cap Reaction Mixture For 100 tests Shortly before use mix 250 ul of L Prepare immediately Catalyst bottle 1 with 11 25 ml of Dye Solution before use The reaction bottle 2 CH mixture cannot be stored For 400 tests Shortly before use mix the total Attention volume 1 ml of one bottle of Catalyst bottle 1 with the total volume 45 ml of one bottle of Dye Solution bottle 2 B Protocol Determining the Optimal Cell Concentration LDH release
6. x 104 P815 test cells well were added to the effector CTL cells The cell mixture was centrifuged and incubated for 4 hr 100 pl of culture supernatant was collected to measure LDH activity 1 5 100 A B 804 3 E lt 10 a z 604 i Ed g g S E 404 S 054 E a g 204 0 0 T T 0 T T 1 10 100 1 10 100 killer target cell ratio killer target cell ratio Figure 5 Measuring the cytolytic activity of allogen stimulated cytotoxicT lymphocytes Panel A Absorbance values Effector cell control O effector test cell mix effector test cell mix minus effector cell control m Panel B Percent cell mediated cytotoxicity calculated as described in Part VI G Notice to Purchaser Clontech products are to be used for research purposes only They may not be used for any other purpose including but not limited to use in drugs in vitro diagnostic purposes therapeutics or in humans Clontech products may not be transferred to third parties resold modified for resale or used to manufacture commercial products or to provide a service to third parties without written approval of Clontech Laboratories Inc Clontech Clontech logo and all other trademarks are the property of Clontech Laboratories Inc unless noted otherwise Clontech is a Takara Bio Company 2007 Clontech Laboratories Inc ATakara Bio Company www clontech com Protocol No PT3947 1 Version No PR6Y2138 16
7. 00 pl freshly prepared Reaction Mixture to a second set of triplicate wells e g the control triplicate Incubate for up to 30 min at room temperature protected from light Measure the absorbance of the samples at 490 or 492 nm using a multiwell plate reader The reference wavelength should be more than 600 nm Compare the average absorbance value of the triplicate containing the test substance with the average absorbance value of the control triplicate Protocol No PT3947 1 Version No 13 PR6Y2138 www clontech com Clontech Laboratories Inc ATakara Bio Company LDH Cytotoxicity Detection Kit Vill References Cook J A amp Mitchell J B 1989 Viability measurements in mammalian cell systems Anal Biochem 179 1 7 Courjault F Leroy D Coquery L amp Toutain H 1993 Platinum complex induced dysfunction of cultured renal proximal tubule cells A comparative study of carboplatin and transplatin with cisplatin Arch Toxicol 67 338 346 Danks A M Hammond D N Wainer B H Van Buskirk R G amp Isaacson R L 1992 Cellular alterations produced by the experimental increase in intracellular calcium and the nature of protective effects from pretreatment with nimodipine Brain Res Mol Brain Res 16 168 172 Decker T amp Lohmann Matthes M L 1988 A quick and simple method for the quantitation of lactate dehydrogenase release in measurements of cellular cytotox icity and tumor necros
8. Lohmann Matthes 1988 Korzeniewski amp Callewaert 1983 High sensitivity Low numbers of dead cells can be detected e g 2 x 10 2 x 10 cells well in a 96 well plate Speed The assay takes only 0 5 1 hr including harvesting supernatants and performing and measuring the enzymatic reaction Large numbers of samples can be processed simultaneously with a multiwell plate reader Simple procedure No need for prelabeling or washing steps Cleanup and waste disposal are simplified since this kit does not employ radioactive isotopes Protocol No PT3947 1 Version No 3 PR6Y2138 www clontech com Clontech Laboratories Inc ATakara Bio Company LDH Cytotoxicity Detection Kit ll List of Components Store unopened LDH Cytotoxicity Detection Kit reagents at 20 C Dissolved catalyst and thawed dye solution can be stored for several weeks at 4 C The LDH Cytotoxicity Detection Kit contains 5 bottles of Catalyst lyophilized and 5 bottles of Dye Solution enough for 2 000 reactions LDH Cytotoxicity Detection Kit Cat No 630117 e 5 bottles Catalyst lyophilized e 5 bottles Dye Solution 45 ml each Supporting Documents e LDH Cytotoxicity Detection Kit User Manual PT3947 1 Visit our web site at www clontech com for a current list of Cell Signaling Apoptosis products Clontech Laboratories Inc www clontech com Protocol No PT3947 1 ATakara Bio Company Version No PR6Y2138 4 LDH Cytotoxicity D
9. a with 1 BSA and the absorbance spectra was measured in the absence and presence of LDH ATakara Bio Company www clontech com Protocol No PT3947 1 Version No PR6Y2138 2 LDH Cytotoxicity Detection Kit l Introduction continued Proven uses of LDH assays include Determination of the cytotoxic potential of compounds Dubar et al 1993 Kondo et al 1993 Murphy et al 1993 Courjault et al 1993 Shrivastava et al 1992 Gelderblom et al 1993 Thomas et al 1993 Sasaki et al 1992 Detection and quantification of cell mediated cytotoxicity induced by cytotoxic T lymphocytes CTL natu ral killer NK cells lymphokine activated killer LAK cells or monocytes Decker amp Lohmann Matthes 1988 Korzeniewski amp Callewaert 1983 Determination of mediator induced cytolysis Decker amp Lohmann Matthes 1988 Measurement of antibody dependent cellular cytotoxicity and complement mediated cytolysis Determination of cell death in bioreactors Goergen et al 1993 Legrand et al 1992 Racher et al 1990 The LDH Cytotoxicity Detection Kit provides several advantages over other cell proliferation assay reagents Safety No radioactive isotopes are used Accuracy Assay results obtained with this kit strongly correlate with the number of damaged cells Further more there is a good correlation between results from the LDH Cytotoxicity Detection Kit assay and the P Cr release assay Decker amp
10. bator e g 37 C 5 CO 90 humidity for the time required to assay the test substance or effector cells 2 24 hr Protocol Incubation time 2 After incubation centrifuge the plate at 250 x g for 10 min lus 45 mi Pane ert 3 Carefully remove 100 pl of supernatant from each well do not disturb the cell pellet and transfer into the corresponding wells of an optically clear 96 well flat bottom plate 4 Add 100 ul freshly prepared Reaction Mixture Part V A to each well and incubate for up to 30 min at room temperature protected from light 5 Measure the absorbance of the samples at 490 or 492 nm using a multiwell plate reader The reference wave length should be more than 600 nm 6 Calculate the percent cytotoxicity according to Protocol F or Protocol G as appropriate F Protocol Calculating Cytotoxicity Due to Soluble Substances Use this formula to calculate cytotoxicity due to the addition of a potentially cytotoxic substance based on the results of Part VI Protocols A B or D 1 Calculate the average absorbance value for each triplicate Subtract the average Background Control value from the average absorbance value for each triplicate to determine absorbance due to cell lysis or other factors in the case of Control triplicates 2 Substitute the resulting values into the following equation Triplicate Absorbance Low Control Cytotoxicity High Control Low Control x 100 G Protocol Cal
11. cells medium 27 1 effector cells medium 0 25 1 effector test cells 28 1 effector test cells 0 25 1 effector cells medium 28 1 effector cells medium 23 1 effector test cells 2 1 effector test cells 24 1 effector test cells 210 1 effector test cells 23 1 effector cells medium 24 1 effector cells medium 2 1 effector cells medium 2 1 effector cells medium 25 1 effector test cells 2 1 effector test cells 25 1 effector cells medium 21 1 effector cells medium ac gt ani fl GD we 2 Low Control High Control D Protocol Assaying Cell Death in Fermentation Cultures 1 Collect samples from the cell culture 0 5 1 ml at regular intervals of 12 24 hr Protocol Culture time then 30 min 2 Centrifuge each sample and carefully remove the culture supernatant without disturbing the cell pellet Note The cell free supernatant can be stored at 4 C for several days without loss of LDH enzyme activity 3 Titrate the culture supernatants with culture medium by serial dilutions into an optically clear 96 well flat bottom plate final volume 100 pl well 4 Proceed to Protocol E step 4 Protocol No PT3947 1 Version No PR6Y2138 11 www clontech com Clontech Laboratories Inc ATakara Bio Company LDH Cytotoxicity Detection Kit VI Measuring Cytotoxicity continued E Protocol Measuring LDH Release 1 Incubate the plate in an incu
12. ction Kit VI Measuring Cytotoxicity continued B Protocol Preparing Adherent Cells to Measure Cytotoxicity Due to Soluble Compounds 1 Plate 100 pl cell suspension in culture medium at the optimal concentration determined in Part V B into wells B1 H6 of a sterile 96 well plate see Table IV cveaiant Note Do not add cells to the Background Control or Substance Control wells A1 A6 see Table IV plus 30 min 2 ee 2 Incubate the cells overnight e g 37 C 5 CO 90 humidity to allow the cells to adhere tightly 3 Immediately before use titrate the test substance in assay medium in wells B1 G6 ofa separate 96 well plate by twofold serial dilution final volume of 200 pl well Note Do not add the test substance to the wells corresponding to the Control wells A1 A6 and H1 H6 see Table IV 4 Remove the culture medium from the adherent cells to remove LDH released from the cells during the overnight incubation Add 100 pl fresh assay medium to each well 5 Transfer 100 pl of the test substance dilutions into the corresponding wells containing adherent cells Note Do not add the test substance to the Control wells A1 A6 and H1 H6 see Table IV 6 Prepare the following controls on the plate see Table IV a Background Control Add 200 pl assay medium to triplicate wells Al A3 b Substance Control I Add 100 pl test substance at the maximum concentration used in the
13. culating Percent Cell Mediated Cytotoxicity Use this formula to calculate cytotoxicity due to effector cells based on the results of Part VI Protocol C 1 Calculate the average absorbance value for each triplicate Subtract the average Background Control value from the average absorbance value for each triplicate to determine absorbance due to cell lysis or other factors in the case of Control triplicates 2 Substitute the resulting values into the following equation using the Effector Test Cell Mix and Effector Cell Control values for a given concentration of effector cells N Effector Test Cell Mix Effector Cell Control Low Control Cytotoxicity r 100 High Control Low Control Clontech Laboratories Inc www clontech com Protocol No PT3947 1 ATakara Bio Company Version No PR6Y2138 12 LDH Cytotoxicity Detection Kit VII Troubleshooting Description of Problem No color reaction Table VI Troubleshooting the LDH Cytotoxicity Detection Kit Explanation Cell concentration may be too low Solution Check cell concentration titrate if necessary Test substance or assay medium inhibit LDH activity Use Substance Control Il to measure test substance and or assay medium for intrinsic LDH inhibition Avoid using media which includes pyruvate an LDH inhibitor Strong color reaction in Low Controls Cell concentration may be too high Check cell concentration titrate if necessary
14. enase release to assess changes in culture viability Cytotechnol 3 301 307 Sasaki T Kawai K Saijo Kurita K amp Ohno T 1992 Detergent cytotoxicity simplified assay of cytolysis by measuring LDH activity Toxicol in Vitro 6 451 457 Shrivastava R Delomenie C Chevalier A John G Ekwall B Walum E amp Massingham R 1992 Comparison of in vivo acute lethal potency and in vitro cytotoxicity of 48 chemicals Cel Biol Toxicol 8 157 170 Szekeres J Pacsa A S amp Pejtsik B 1981 Measurement of lymphocyte cytotoxicity by assessing endogenous alkaline phosphatase activity of the target cells J Immunol Meth 40 151 154 Thomas J P Geiger P G amp Girotti A W 1993 Lethal damage to endothelial cells by oxidized low density lipoprotein role of selenoperoxidases in cytoprotection against lipid hydroperoxide and iron mediated reactions J Lipid Res 34 479 490 Yuhas J M Toya R E amp Pazmino N H 1974 Neuraminidase and cell viability failure to detect cytotoxic effects with dye exclusion techniques J Nat Cancer Inst 53 465 468 Clontech Laboratories Inc ATakara Bio Company www clontech com Protocol No PT3947 1 Version No PR6Y2138 14 LDH Cytotoxicity Detection Kit Appendix A Examples of LDH Cytotoxicity Detection Kit Results A Optimizing Cell Concentration K562 cells were titrated in 96 well plates at the concentrations indicated on the horizontal axis of Figure 3 Cu
15. etection Kit Ill Additional Materials Required The following materials are required but not supplied Incubator 37 C Centrifuge with rotor for 96 well plates Multiwell plate reader with a 490 or 492 nm filter Ifa reference wavelength is to be subtracted an additional filter above 600 nm is required Multichannel pipettor 100 pl Sterile pipette tips 96 well plates sterilized cell culture quality for measurements of cell mediated lysis and for the analysis of cytotoxic compounds e For suspension cells round or V bottom 96 well plates e For adherent cells flat bottom 96 well plates e For color development in all assays optically clear flat bottom 96 well plates Assay Medium e g medium containing 1 serum or 1 bovine serum albumin Both human and animal sera contain LDH which may increase the background absorbance of the assay Therefore we recommend performing the assay in the presence of low serum concentrations e g 1 or replacing the serum with 1 bovine serum albumin w v Triton X 100 lysing solution 2 Triton X 100 in assay medium The maximum LDH activity is deter mined by lysing the cells with Triton X 100 final concentration 1 At this concentration Triton X 100 does not interfere with LDH activity HCI stop solution 1 N The reaction product can be measured without adding a stop solution Alternatively the enzyme reaction can be stopped by adding 50 pl of 1 N HCI to each well final co
16. experiment and 100 pl well assay medium to triplicate wells A4 A6 c Low Control for spontaneous LDH release Add 200 pl assay medium to triplicate wells H1 H3 d High Control for maximum LDH release Add 100 pl assay medium and 100 pl Triton X 100 solution to triplicate wells H4 H6 7 Proceed to Protocol E Table IV Plate Layout to Measure Cytotoxicity Due to Soluble Compounds tt 2 8 4 5 6 7 8 9 10 11 12 A Background Control Substance Control B 1 1 test substance cells 26 1 test substance cells G 0 5 1 test substance cells 27 1 test substance cells D 0 25 1 test substance cells 28 1 test substance cells E 23 1 test substance cells 2 1 test substance cells F 24 1 test substance cells 210 1 test substance cells G 25 1 test substance cells 2 1 test substance cells H Low Control High Control Table IV 96 well plate layout to assay cytotoxicity Columns 1 6 Note that a second test substance could be assayed in columns 7 12 Clontech Laboratories Inc www clontech com Protocol No PT3947 1 ATakara Bio Company Version No PR6Y2138 10 LDH Cytotoxicity Detection Kit VI Measuring Cytotoxicity continued C Protocol Preparing Samples to Measure Cell Mediated Cytotoxicity 1 Table V outlines the plate setup the 96 well plate is divided in half with identical dilutions of the effector cell suspension in wells B1 G6 and B7 G12 Titrate the effector cells in triplicate by twofold serial dilu Protocol 30 min tions in assay med
17. free culture supernatant is collected and incubated with the reaction mixture from the kit LDH activity is determined by a colorimetric assay In the first step NAD is reduced to NADH H by the LDH catalyzed conversion of lactate to pyruvate In the second step a catalyst included in the reaction mixture diaphorase transfers H H from NADH H to the tetrazolium salt INT which is reduced to a formazan dye Figure 1 LDH Lactic acid gt Pyruvic acid NAD NADH H Eterna ec Tetrazolium red Diaphorase yellow Figure 1 A two step enzymatic reaction quantifies cell lysis and cell death An increase in the number of dead or plasma membrane damaged cells leads to increased LDH activity in the culture supernatant which directly correlates with the amount of formazan produced in a defined time period Therefore the amount of dye produced is proportional to the number of lysed dead or plasma membrane damaged cells The red formazan dye product is water soluble and shows a broad absorbance maximum around 500 nm while the tetrazolium salt INT shows little absorbance at this wavelength Figure 2 3 0 2 575 absorbance gt N o Oo fi I ess fo L 0 54 nT 0 T T T T E 400 450 500 550 600 650 700 wavelength nm Figure 2 Absorbance spectra of the LDH Cytotoxicity Detection Kit Reaction Mixture The reaction mixture from the LDH Cytotoxicity Detection Kit was added to RPMI 1640 medi
18. is factor TNF activity J Immunol Meth 115 61 69 Dubar V Gosset P Aerts C Voisin C Wallaert B amp Tonnel A B 1993 Jn vitro acute effects of tobacco smoke on tumor necrosis factor alpha and interleukin G production by alveolar macrophages Exp Lung Res 19 345 359 Gelderblom W C Cawood M E Snyman S D Vleggaar R amp Marasas W F 1993 Structure activity relationships of fumonisins in short term carcinogenesis and cytotoxicity assays Food Chem Toxicol 31 407 414 Goergen J L Marc A amp Engasser J M 1993 Determination of cell lysis and death kinetics in continuous hybridoma cultures from the measurement of lactate dehydrogenase release Cytotechnol 11 189 195 Jones K H amp Senft J A 1985 An improved method to determine cell viability by simultaneous staining with fluorescein diacetate propidium iodide J Histo chem Cytochem 33 77 79 Kolber M A Quinones R R Gress R E amp Henkart P A 1988 Measurement of cytotoxicity by target cell release and retention of the fluorescent dye bis carboxy ethyl carboxyfluorescein BCECEF J Immunol Meth 108 255 264 Kondo T Takahashi S Sato H Yamada M Kikuchi T amp Furuya K 1993 Cytotoxicity of size density fractionated coal fly ash in rat alveolar macrophages cultured in vitro Toxicol in Vitro 7 61 67 Korzeniewski C amp Callewaert D M 1983 An enzyme release assay for natural cytotoxicity J Immun
19. ium final volume 100 pl well using a multichannel pipette Note Do not add effector cells to the Control wells Rows A amp H see Table V 2 Wash the test cells in assay medium and dilute them to their optimal concentration Part V B 3 Add 100 pl well test cell suspension to the effector cell dilutions in wells B1 G6 see Table V Note Do not add test cells to the Control wells A1 A6 and H1 H6 see Table V or to the effector cell only wells B7 G12 4 Prepare the following controls on the plate see Table V a Background Control Add 200 pl assay medium to triplicate wells Al A3 b Low Control for spontaneous LDH release Add 100 pl test cell suspension and 100 pl assay medium to triplicate wells H1 H3 c High Control for maximum LDH release Add 100 pl test cell suspension and 100 pl Triton X 100 solu tion to triplicate wells H4 H6 d Spontaneous LDH release for each effector cell concentration Add 100 yl assay medium to wells B7 G12 Note Spontaneous LDH release must be determined for each effector cell concentration used in the assay 5 Proceed to Protocol E tv 2 3 4 5 6 Table V Plate Layout to Measure Cell Mediated Cytotoxicity 7 8 9 10 11 12 Background Control 1 1 effector test cells 26 1 effector test cells 1 1 effector cells medium 28 1 effector cells medium 0 5 1 effector test cells 27 1 effector test cells 0 5 1 effector
20. kara Bio Company 1 Clontech Laboratories Inc Introduction LDH Cytotoxicity Detection Kit Cell death is typically assayed by quantifying plasma membrane damage Many standard methods are based on the uptake or exclusion of vital dyes Cook amp Mitchell 1989 Yuhas et al 1974 Parks et al 1979 Jones amp Senft 1985 or on the release of radioactive isotopes fluorescent dyes or calcein AM from prelabeled target cells Oldham et al 1977 Leibold amp Bridge 1979 Kolber et al 1988 Danks et al 1992 Other assays measure cytoplasmic enzymes released by damaged cells where the amount of enzyme activity detected in the culture supernatant correlates with the proportion of lysed cells Decker amp Lohmann Mathes 1988 Szekeres et al 1981 Masanet et al 1988 Martin amp Clynes 1991 Enzyme release assays have been described for alkaline and acid phosphatase glutamate oxalacetate transaminase glutamate pyruvate transaminase and arginosuccinate lyase However their use has been hampered by the low amount of these enzymes present in many cells and by the elaborate kinetic assays required to quantitate most enzyme activities In contrast lactate dehydrogenase LDH is a stable cytoplasmic enzyme which is present in all cells When the plasma membrane is damaged LDH is rapidly released into the culture supernatant The LDH Cytotoxicity Detection Kit provides a simple and precise colorimetric method to measure LDH activity Cell
21. lture medium 0 was added to measure spontaneous LDH release Low Control and Triton X 100 was added final concentration 1 to measure the maximum LDH release High Control The optimal cell concentration in this experiment was between 1 x 104 and 1 x 10 cells well 3 0 2 5 ODg20nm N te oa absorbance ODag2nm gt Oo 0 5 cells well Figure 3 Optimizing K562 cell concentration B Measuring Detergent Cytotoxicity Three detergents Synperonic F68 m Triton X 100 a and Nonident P40 Y were titrated with culture medium in a 96 well plate to the final concentrations indicated on the horizontal axis of Figure 4 Subsequently P815 cells were added final concentration 1 x 10 cells well The cells were incubated for 18 hr and LDH release was measured 1 0 ji Ass0nm So a absorbance Agganm 104 103 10 detergent conc Figure 4 Measuring detergent cytotoxicity Synperonic F68 m Triton X 100 A and Nonident P40 Y Protocol No PT3947 1 www clontech com Clontech Laboratories Inc Version No PR6Y2138 ATakara Bio Company 15 LDH Cytotoxicity Detection Kit Appendix A Examples of LDH Cytotoxicity Detection Kit Results continued C Measuring Cell Mediated Toxicity Spleen cells of C57 B1 6 mice H 2b were stimulated in vitro with P815 cells H 2d Viable cytotoxic T lymphocytes CTLs were purified by ficoll density gradient washed and titrated in a 96 well plate 1
22. ncentra tion 0 2 N HCl LDH standard solution If the released LDH activity must be calculated as Units ml instead of percent cyto toxicity we recommend that you prepare a standard curve using an LDH standard solution Microscope Hemacytometer Protocol No PT3947 1 Version No 5 PR6Y2138 www clontech com Clontech Laboratories Inc ATakara Bio Company LDH Cytotoxicity Detection Kit IV LDH Cytotoxicity Detection Kit Protocol Overview PLEASE READ ALL PROTOCOLS IN THEIR ENTIRETY BEFORE BEGINNING Successfully implementing the LDH Cytotoxicity Detection Kit consists of performing the steps listed below all of which are included in this user manual A Protocol Overview The LDH Cytotoxicity Detection Kit is a simple colorimetric assay to quantitate cytotoxicity cytolysis and is based on the measurement of LDH activity released from damaged cells into the supernatant Table I The cell free culture supernatant is collected and incubated with the Reaction Mixture to determine LDH activity and quantify cell death Specific protocols are included for four different types of experiments e Measuring cytotoxicity caused by soluble compounds for suspended cells e Measuring cytotoxicity caused by soluble compounds for adherent cells e Measuring cell mediated cytotoxicity caused by effector cells e Measuring cell death in fermentation cultures Table I Protocol Overview Step Procedure Time Optimizing E
23. ncubate at room temperature for 30 min protected from light Measure the absorbance of the samples at 490 or 492 nm The reference wavelength should be greater than 600 nm 10 Calculate the average absorbance value of each triplicate and subtract the average value for the Background Control from the triplicate average Compare the Low and High Control values at each cell concentration The cell concentration with the greatest difference between the absorbance of the Low and High Controls is the optimal cell concentration for your experiment see Appendix A part A for an example Clontech Laboratories Inc ATakara Bio Company www clontech com Protocol No PT3947 1 Version No PR6Y2138 8 LDH Cytotoxicity Detection Kit VI Measuring Cytotoxicity Once you have optimized the cell concentration for your experiment Part V B the assay steps are similar in each case although the sample preparation varies Protocols A D below In brief the assay steps are Protocol 30 min 1 2 Bs 4 5 6 Incubate the optimal concentration of cells in a 96 well plate with the test substance or effector cells at varying concentrations Centrifuge the 96 well plate Transfer cell free supernatant to an optically clear flat bottom 96 well plate Add freshly prepared Reaction Mixture and incubate to allow the enzymatic reaction to take place Figure 1 Optional Add 1 N HCI not included in the kit to stop the reaction
24. ol Meth 64 313 320 Legrand C Bour J M Jacob C Capiaumont J Martial A Marc A Wudtke M Kretzmer G Demangel C Duval D et al amp Hache J 1992 Lactate dehy drogenase LDH activity of the cultured eukaryotic cells as marker of the number of dead cells in the medium corrected J Biotechnol 25 231 243 Erratum in J Biotechnol 1993 31 234 Leibold W amp Bridge S 1979 Se release a short and long term assay system for cellular cytotoxicity Z Immunitatsforschung Immunobiol 155 287 311 Martin A amp Clynes M 1991 Acid phosphatase endpoint for in vitro toxicity tests In Vitro Cell Dev Biol 27A 183 184 Masanet J Gomez Lechon M J amp Castell J V 1988 Hepatic toxicity of paraquat in primary cultures of rat hepatocytes Toxicol in Vitro 2 275 282 Murphy E J Roberts E amp Horrocks L A 1993 Aluminum silicate toxicity in cell cultures Neurosci 55 597 605 Oldham R K Ortaldo J R Holden H T amp Herberman R B 1977 Direct comparison of three isotopic release microtoxicity assays as measures of cell mediated immunity to Gross virus induced lymphomas in rats J Natl Cancer Inst 58 1061 1067 Parks D R Bryan V M Oi V T amp Herzenberg L A 1979 Antigen specific identification and cloning of hybridomas with a fluorescence activated cell sorter Proc Natl Acad Sci USA 76 1962 1966 Racher A J Looby D amp Griffiths J B 1990 Use of lactate dehydrog
25. ton X 100 F 24 1 cells medium 21 1 cells medium 24 1 cells Triton X 100 21 1 cells Triton X 100 G 25 1 cells medium 212 1 cells medium 25 1 cells Triton X 100 212 1 cells Triton X 100 H 26 1 cells medium 218 1 cells medium 26 1 cells Triton X 100 213 1 cells Triton X 100 Low Controls High Controls Protocol No PT3947 1 www clontech com Clontech Laboratories Inc Version No PR6Y2138 ATakara Bio Company 7 LDH Cytotoxicity Detection Kit V Sample Preparation and Experiment Setup continued 4 Prepare the following controls a Background control Add 100 pl assay medium to wells Al A3 b Low Controls for spontaneous LDH release Add 100 pl well assay medium to Rows B H Columns 1 6 c High Controls for maximal LDH release Add 100 pl well Triton X 100 to Rows B H Columns 7 12 Incubate the plate in a humidified atmosphere 37 C 5 CO 90 humidity for the time required to as say the test substances in your experiment typically in the range of 2 24 hr Note Each well contains a total volume of 200 ul 100 ul of assay medium plus either 100 ul of cell suspension steps 2 3 or 100 ul of Control substance step 4 Centrifuge the plate at 250 x g for 10 min Remove 100 pl well supernatant carefully without disturbing the cell pellet Transfer the supernatant into the corresponding wells of an optically clear 96 well flat bottom plate Add 100 pl Reaction Mixture freshly prepared Part V A to each well and i
26. varies among cell types Therefore you must determine the optimal cell concentration where the differ ence between the High and Low Controls is at a maximum for each cell type The optimal cell concentration for most Protocol cell lines is between 5 x 10 and 2 x 104 cells well in 200 pl of media 2 5 x 104 1 x 10 cells ml Incubation time plus 1 hr 1 Fill the entire 96 well plate with 100 pl well assay medium 2 Wash the cells with assay medium and then adjust the cell suspension to a concentration of 2 x 10 cells ml with assay medium 3 Table III outlines the plate setup the 96 well plate is divided in half with identical dilutions of the cell suspension in wells B1 H6 and B7 H12 Titrate the cells by twofold serial dilutions in assay medium using a multichannel pipette a Transfer 100 pl well cell suspension into wells B1 B3 and B7 B9 Dilution 1 b Transfer 100 pl of the diluted cell suspension from these wells into C1 C3 and C7 C9 Dilution 2 Repeat this step 12 times to prepare all the cell suspension dilutions Table Ill Plate Layout to Optimize Cell Concentration A Background Control B 1 1 cells medium 27 1 cells medium 1 1 cells Triton X 100 27 1 cells Triton X 100 G 0 5 1 cells medium 28 1 cells medium 0 5 1 cells Triton X 100 28 1 cells Triton X 100 D 0 25 1 cells medium 2 1 cells medium 0 25 1 cells Triton X 100 22 1 cells Triton X 100 E 23 1 cells medium 210 1 cells medium 23 1 cells Triton X 100 210 1 cells Tri
27. vscvesssneresaessvabedbuegsspvaetoensssevedveve 15 C Measuring Gell Mediated Toxicity sess sess cesssshsexstsccvescuevsee havea venstichanteetant EEEE EN RE 16 List of Figures Figure 1 A two step enzymatic reaction quantifies cell lysis and cell death 0 eeeeeeeneeeeeeeeneeeeeeeeees 2 Figure 2 Absorbance spectra of the LDH Cytotoxicity Detection Kit Reaction Mixture cece 2 Figure 3 Optimizing cell concentrations sicictsnesssecsssdscscadsneesicniestesevestebetevessessorsngevbaaodawnse ens EEan 15 Figure 4 Measuring detergent Cytotoxicity sesccsssesssecsesessecesesescceecssnseesecasaesesenaeseaseceasecateeeesesaesesees 15 Figure 5 Measuring the cytolytic activity of allogen stimulated cytotoxic T lymphocytes eee 16 List of Tables Table i Protocol Ove mi W cscs series saraiisianenane iii Gis REEE ninine aie Gia ose 6 Table Ti Working SOMOS sasise aneii eioten eiTe eva Ne EERE e IETEN TOTENS ENAN 7 Table II Plate Layout to Optimize Cell Concentratioiiisrisos isss setreeren toese ienasi ieena 7 Table IV Plate Layout to Measure Cytotoxicity Due to Soluble Compounds eeeeeeesetetereneeeeeeneees 10 Table V Plate Layout to Measure Cell Mediated Cytotoxicity cccccceeseccssescseeeceeeceeeeceeeeeseeseseeeeeeneeeees 11 Table VI Troubleshooting the LDH Cytotoxicity Detection Kit ccc ceeceseeescnseeeeseeeeeseesrseeneeaneeneaes 13 Protocol No PT3947 1 www clontech com Clontech Laboratories Inc Version No PR6Y2138 ATa
28. xperimental Conditions ile Prepare working solutions 15 min 2 Optimize cell concentration Incubation time approx 45 min Performing the Experiment th Incubate cells with test substance or cytotoxic effector cells 2 24 hr 2 Prepare working solutions 15 min 3 Centrifuge the 96 well plate containing the cells 10 min 4 Transfer cell free culture supernatant to optically clear flat bottom 96 well plate 5 Incubate the supernatant with freshly prepared Reaction Mixture containing the approx 10 30 min tetrazolium salt INT gt Optional Stop the reaction by adding 1 N HCI to each well Measure absorbance at 490 or 492 nm reference wavelength 690 nm and calcu late percent cytotoxicity B Experiment Controls To calculate the percent cytotoxicity you must perform the following three controls in every experiment e Background Control Measures the LDH activity present in the assay medium The background absorbance must be subtracted from all other absorbance measurements e Low Control Measures the level of spontaneous LDH release from untreated cells e High Control Measures the maximum LDH activity that can be released from the 100 dead cells in response to Triton X 100 In addition the following two controls may be required to perform your experiment or for troubleshooting e Substance Control I Measures the LDH activity contained in the test substance Alternatively if
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