MTT升级版细胞计数试剂盒CCK8说明书

Revised August 16, 2002 Cell Counting Kit-8 Technical Manual Contents General Information ........................................................................................... 1 Advantages ........................................................................................................ 1 Storage .............................................................................................................. 2 How to Use Cell Counting Kit-8 .......................................................................... 2 Required Equipments and Materials ........................................................ 2 Protocol .................................................................................................... 2 Cell Proliferation Assay ...................................................................... 2 Cytotoxicity Assay .............................................................................. 2 Background Control ........................................................................................... 2 Precautions ........................................................................................................ 3 Frequently Asked Questions .............................................................................. 3 References ........................................................................................................ 3 Appendix ............................................................................................................ 4 Cytotoxicity Assay Data ...................................................................... 4 Product Code and Price ..................................................................................... 4 Related Products ............................................................................................... 4 General Protocol at a Glance ............................................................................. 5 For the most up-to-date technical manual, access www.dojindo.com/tm Product Code: CK04-11 (1000 tests) CK04-13 (3000 tests) Cell Proliferation Assay and Cytotoxicity Assay 300 400 500 600 700 0 1.0 2.0 3.0 Wavelength/ nm - 1 - GENERAL INFORMATION Cell Counting Kit-8 (CCK-8) allows very convenient assays by utilizing Dojindo’s highly water-soluble tetrazolium salt. WST-8 (2-(2-methoxy-4-nitrophenyl)-3- (4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt)* produces a water-soluble formazan dye upon reduction in the presence of an electron carrier as shown in Figure 1. Cell Counting Kit-8 is a one-bottle solution; no premixing of components is required. Cell Counting Kit- 8, being non-radioactive, allows sensitive colorimetric assays for the determination of the number of viable cells in cell proliferation and cytotoxicity assays. WST-8 is reduced by dehydrogenases in cells to give a yellow- colored product (formazan), which is soluble in the tissue culture medium. The amount of the formazan dye generated by the activity of dehydrogenases in cells is directly proportional to the number of living cells. The detection sensitivity of CCK-8 is higher than other tetrazolium salts such as MTT, XTT, MTS or WST-1. *Patent No. WO97/38985 Figure 1. Structures of WST-8 and WST-8 formazan Figure 2 shows the absorption spectrum of WST-8 formazan. Since the absorbance at 460 nm is proportional to the number of viable cells in the medium, the viable cell number can be determined using the absorbance value of a previously prepared calibration curve. Figure 2. Absorption spectrum of WST-8 formazan As shown in Figure 3, the absorbance of the CCK-8 assay solution is higher than that of MTT and other tetrazolium salts that produce water-soluble formazan dyes. Figure 4 shows that the cell proliferation assay using CCK-8 correlates well with the [3H]-thymidine incorporation assay. Thus, the CCK-8 assay can also be substituted for the [3H]- thymidine incorporation assay. Figure 3: Cell proliferation assay using CCK-8 and other reagents Culture medium: MEM, 10% FCS, L-glutamine (HeLa) RPMI1640, 10% FCS, L-glutamine (HL60) Incubation: 37 °C, 5% CO2, 2 hrs (HeLa) 37 °C, 5% CO2, 3 hrs (HL60) Detection : CCK-8 ( ): 450 nm, reference: 650 nm XTT ( ): 450 nm, reference: 650 nm MTS ( ): 490 nm, reference: 650 nm MTT ( ): 570 nm, reference: 650 nm Figure 4: Correlation between [3H]-thymidine incorporation assay and CCK-8 assay. Cell line: HeLa, HL60 Culture medium: MEM, 10% FCS (HeLa) RPMI1640, 10% FCS (HL60) Reagent: [3H]-thymidine: 37 KBq/well CCK-8: 10 µl/well Incubation: [3H]-thymidine assay: 4 hrs CCK-8 assay: 3 hrs ADVANTAGES - One-bottle, ready-to-use solution - No organic solvent or isotope required - No harvesting, no washing and no solubilization steps - More sensitive than MTT, XTT, MTS or WST-1 N N N N O2N SO3- SO3- O2N OCH3 + Na+ N N N N O2N SO3- SO3- O2N OCH3 H WST-8 Formazan !!!!! """"" !!!!! """"" 0.0 0.5 1.0 1.5 2.0 0.0 0.5 1.0 1.5 2.0 [ H]-Thymidine x10 dpm HeLa Cell r=0.999 0.0 0.1 0.2 0.3 0.0 1.0 2.0 3.0 [ H]-Thymidine x10 dpm HL60 Cell r=0.996 -5-5 33 0 1 2 3 0 5 10 15 20 25 Number of cells x10 /well 0 0.1 0.2 0.3 0 5 10 15 20 25 Number of cells x10 /well HeLa Cell HL60 Cell -3 -3 0 50 100 Concentration of chemical (mM) MMC SDS 10-610-510-410-310-210-1 1 1010 -6 10-5 10-4 10-3 10-2 10-1 1 10 Concentration of chemical (mM) O O N H2N H2N O O O NH2 CH3 NH mitomycin-C (MMC) CH3(CH2)11-OSO3Na Dodecylsulfate, sodium salt (SDS) - 2 - STORAGE As shown in Table 1, CCK-8 is stable for over 1 year at -20 °C and 3 months at 4 °C with protection from light. However, repeated thawing and freezing causes an increase in the background, which interferes with the assay. To avoid repeated thawing and freezing, keep the kit at 4 oC if it is frequently used. Table 1. Blank absorbances at 460 nm of CCK-8 at -20 °C or 4 °C storage. Storage / month Absorbance at 460 nm - 20 °C 4 °C 0 0.46 0.46 0.5 0.46 0.46 1 0.46 0.46 3 0.46 0.46 6 0.46 0.51 12 0.46 0.59 HOW TO USE CELL COUNTING KIT-8 HOW TO USE TOTAL GLUTATHIONE ASSAY KIT 1. Required Equipments and Materials • plate reader (450 nm filter) • 96-well plate • 10 µl, 100-200 µl and multi-channel pipettes • CO2 incubator 2. Protocol Cell Proliferation Assay 1) Inoculate cell suspension (100 µl/well) in a 96-well plate. Also prepare wells that contain known numbers of viable cells (to be used to create a calibration curve in step 5). Pre-incubate the plate in an incubator (humidified atmosphere; e.g., at 37 °C, 5% CO2). 2) Thaw the frozen CCK-8 on the bench top or in a water bath at 37 °C. It takes about 30 minutes on the bench top at 25 °C or 5 minutes in the water bath at 37 °C. 3) Add 10 µl of the CCK-8 solution to each well of the plate. Be careful not to introduce bubbles to the wells since the bubbles interfere with the O.D. reading. 4) Incubate the plate for 1-4 hours in the incubator. 5) Measure the absorbance at 450 nm using a microplate reader. Prepare a calibration curve using the data obtained from the wells that contain known numbers of viable cells. To measure the absorbance later, add 10 µl of 1% w/v SDS to each well, cover the plate and store it with protection from light at room temperature. No absorbance change should be observed for 48 hours. Cytotoxicity Assay 1) Dispense 100 µl of cell suspension (5000 cells/ well) in a 96-well plate. 2) Pre-incubate the plate for 24 hours in an incubator (humidified atmosphere; e.g. at 37 °C, 5% CO2). 3) Add 10 µl of various concentrations of toxicant into the culture media in the plate. 4) Incubate the plate for 48 hours in the incubator. 5) Thaw the frozen CCK-8 on the bench top or in a water bath at 37 °C. It takes about 30 minutes on the bench top at 25 °C or 5 minutes in the water bath at 37 °C. 6) Add 10 µl of CCK-8 solution to each well of the plate. Be careful not to introduce bubbles to the wells since the bubbles interfere with the O.D. reading. 7) Incubate the plate for 1-4 hours in the incubator. Measure the absorbance at 450 nm using a plate reader. To measure the absorbance later, add 10 µl of 1% w/v SDS to each well, cover the plate and store it with protection from light at room temperature. No absorbance change should be observed for 48 hours. Figure 5. Toxicological test of chemicals using CCK-8 Cell line: HeLa Culture medium: MEM, 10% FCS, L-glutamine Chemicals: Mitomycin-C (MMC) Dodecylsulfate, sodium salt (SDS) Incubation: 37 °C, 5% CO2, 2hrs Detection: 450 nm, reference: 650 nm BACKGROUND CONTROL 1. Slight spontaneous absorbance around 460 nm occurs in the culture medium incubated with CCK-8. This background absorbance depends on the culture medium, pH, incubation time and length of exposure to light. Typical background absorbance after 2 hours incubation is 0.1 - 0.2 absorbance units. Prepare one or more control wells without cells, and subtract the average absorbance of the control wells from that of the other wells. 2. During a five-hour experiment, the absorbance of the CCK-8 solution does not increase at room temperature. - 3 - PRECAUTIONS 1. Since the CCK-8 assay is based on the dehydrogenase activity detection in viable cells, conditions or chemicals that affect dehydrogenase activity in viable cells may cause discrepancy between the actual viable cell number and the cell number determined using the CCK-8 assay. 2. WST-8 may react with reducing agents to generate WST-8 formazan. Please check the background O.D. if reducing agents are used in cytotoxicity assays or cell proliferation assays. 3. Be careful not to introduce bubbles to the wells since the bubbles interfere with the O.D. reading. 4. Phenol red containing culture media can be used with this kit for cell viability assays. 5. A membrane filtration is recommended for the sterilization of the CCK-8 solution, if necessary. 6. The incubation time varies by cell types and/or cell numbers in a well. Generally, leukocytes give weak coloration, thus a long incubation time (up to four hours) or a large number of cells (~105 cells/well) may be necessary. 7. Since the cytotoxicity of this kit is very low, further color development is possible after reading the absorbance. 8. Neutral red or crystal violet can be used after the CCK-8 assay. 9. Measure the reference wavelength at 600 nm or higher if there is a high turbidity in the cell suspension. 1. How many cells should there be in a well? For adhesive cells, at least 1000 cells are necessary per well (100 µl medium) when using the kit's standard 96-well plate. For leukocytes, at least 2500 cells are necessary per well (100 µl medium) because of the low sensitivity. The recommended maximum number of cells per well for the 96-well plate is 25000. If 24- well plate or 6-well plate is used for this assay, please calculate the number of cells per well accordingly, and adjust the volume of the CCK-8 solution in a well to 10% of the total volume. 2. Does CCK-8 stain viable cells? No, it does not stain viable cells because the water-soluble tetrazolium salt-8 (WST-8) is used in the CCK-8 solution. The electron mediator, 1-Methoxy PMS, receives electrons from a viable cell and transfers the electron to WST-8 in the culture medium. Since its formazan dye is also highly water-soluble, CCK-8 can not be utilized for a cell staining purpose. REFERENCES FREQUENTLY ASKED QUESTIONS 3. How stable is CCK-8? CCK-8 is stable over one year at -20 oC, 3 months at 4 oC and 1 week at room temperature. CCK-8 is stable over 2 days even at 60 oC as long as the CCK-8 solution keeps its original red color and does not turn orange. 4. Does phenol red affect the assay? No. The absorption value of phenol red in a culture medium can be removed by subtracting the absorption value of a blank solution from the absorption value of each well. Therefore, a phenol red containing medium is usable for the CCK-8 assay. 5. Is there a correlation between CCK-8 and the Thymidine incorporation assay? Yes. For correlation graphs, see page 1. Please note that since CCK-8 uses a different assaying mechanism from that of the Thymidine assay as described on page 1, the CCK-8 and Thymidine assay results may differ. 6. Is CCK-8 toxic to cells? The toxicity of CCK-8 is so low that, after the CCK-8 assay is completed, the same cells can be used for other cell proliferation assays such as the crystal violet assay, neutral red assay or DNA fluorometric assay. 7. Can I use CCK-8 for 384-well plates or buy it in bulk quantity? CCK-8 can be used for 384-well plates. Please dilute the CCK-8 solution using PBS since the required volume of the CCK-8 solution is 5 µl per well in case of the 384-well plate. We also offer CCK-8 for HTS (high throughput screening) and CCK-8 in bulk quantities (Product Code: CK04-20). Please contact us at info@dojindo.com or 1-877-987-2667 for more information. 1. M. Ishiyama, H. Tominaga, M. Shiga, K. Sasamoto, Y. Ohkura and K. Ueno, Biol. Pharm. Bull. 19, 1518 (1996). 2. M. Ishiyama, Y. Miyazono, K. Sasamoto, Y. Ohkura, and K. Ueno, Talanta, 44, 1299 (1997). 3. H. Tominaga, M. Ishiyama, F. Ohseto, K. Sasamoto, T. Hamamoto, K. Suzuki, M. Watanabe, Anal. Commun., 36, 47 (1999). 4. I. Isobe, M. Michikawa, K. Yanagisawa, Neurosci. Lett., 266, 129 (1999). 5. M. Matsuoka, B. Winsprivono, H. Igisu, Biochem. Pharmacol., 59, 1573 (2000). 6. Y. Bai, A. Suzuki, M. Sagai, Free Radic. Biol. Med., 30, 555 (2001). 7. K. Yoshimura, A. Tanimoto, T. Abe, M. Ogawa, T. Yutsudo, M. Kashimura, S. Yoshida, J. Soc. Gynecol. Investig., 1, 22 (2002). - 4 - APPENDIX Cytotoxicity Assay Data Table 2. The LD50 values of anti-tumor agents to human cancer cell lines using CCK-8 and MTT. Cell lines Anti-tumor CCK-8 MTT agents LD50 (µg/ml) LD50 (µg/ml) HE49 Bleomycin 154.59 157.34 Cisplatin 1.55 1.61 Adriamycin 0.41 0.47 MMC 0.05 0.03 Etopside 13.01 13.18 5-FU 0.12 0.37 MKN-28 Bleomycin 151.18 139.51 Cisplatin 0.57 0.60 Adriamycin 0.11 0.13 MMC 0.02 0.02 Etopside 4.11 4.63 5-FU 0.05 0.05 IMR-32 Bleomycin 314.15 207.00 Cisplatin 5.65 5.32 Adriamycin 2.27 0.33 MMC 0.10 0.10 Etopside 9.63 9.71 5-FU 0.04 0.05 H1299 Bleomycin 406.65 1057.12 Cisplatin 0.51 0.72 Adriamycin 0.17 0.14 MMC 0.04 0.06 Etopside 14.63 25.96 5-FU 0.08 0.05 HL60 Bleomycin 280.74 190.84 Cisplatin 1.54 1.00 Adriamycin 0.19 0.24 MMC 0.02 0.03 Etopside 12.23 11.32 5-FU 0.06 0.07 MOLT-4 Bleomycin 218.84 187.26 Cisplatin 0.94 1.04 Adriamycin 0.27 0.24 MMC 0.01 0.01 Etopside 3.87 4.27 5-FU 0.11 0.11 KC12 Bleomycin 739.05 880.56 Cisplatin 3.59 2.98 Adriamycin 0.37 0.48 MMC 0.15 0.11 Etopside 52.87 53.31 5-FU 0.20 0.19 HeLa Bleomycin 306.89 379.24 Cisplatin 15.22 11.52 Adriamycin 1.56 1.98 MMC 0.18 0.14 Etopside 79.16 58.37 5-FU 0.71 0.78 Cell lines: HE49: human normal embryo MKN-28: human gastric cancer IMR-32: human neuroblastoma H1299: human lung cancer HL60: human acute promyelonic leukemia MOLT-4: human acute lymphoblastic leukemia KC12: human renal cancer HeLa: human cervical cancer Anti-tumor agents: MMC (Mitomycin C), 5-FU (5-Fluoro- uracil) PRODUCT CODE AND PRICE Product Units Product Code Price ($) CCK-8* 1000 tests CK04-11 150.00 CCK-8* 3000 tests CK04-13 300.00 CCK-8* 20000 tests or more CK04-20 request * CCK-8: Cell Counting Kit-8 RELATED PRODUCTS Product Units Product Code Price ($) CCK-F* 500 tests CK06-10 98.00 * Cell Counting Kit-F High sensitivity fluorometric assay for the determination of viable cell number as low as 50 cells / well (100 µl volume / well). For technical questions, please contact us at info@dojindo.com or 1-877-987-2667 (toll free). - U.S.A. - Dojindo Molecular Technologies, Inc. 211 Perry Parkway, Suite 5 Gaithersburg, MD 20877 Phone: 301-987-2667 Fax: 301-987-2687 E-mail: info@dojindo.com Web site: www.dojindo.com - JAPAN - Dojindo Laboratories Kumamoto Techno Research Park 2025-5 Tabaru, Mashiki-machi, Kamimashiki-gun Kumamoto 861-2202, JAPAN Phone: +81-96-286-1515 Fax: +81-96-286-1525 E-mail: info@dojindo.co.jp Web site: www.dojindo.co.jp Add 10 µla) of Cell Counting Kit-8 (CCK-8) solution to each well in an assay plateb). Incubate the plate in a CO2 incubator for 1 - 4 hours.c) Put the plate in a microplate reader, and read the O.D. at 450 nm. Determine the viable cell numbers in sample media using the calibration curve (derived from the cell suspensions containing known numbers of viable cells), or determine LD50 of toxicant used. a) use 1/10 volume of CCK-8 solution to each cell culture medium in a well (i.e. 10 µl CCK-8 solution for 100 µl cell culture medium). b) be careful not to introduce bubbles to the wells since the bubbles interfere with the O.D. reading. 1- 4 hours Step 1) Step 2) Step 3) Step 4) Add 10 µl of 1% w/v SDS solution to each well, and store the plate at room temperature with protection from light.d) c) adhesive cells: 1 - 2 hours incubation non-adhesive cells (leukocytes): 3 - 4 hours incubation Skip Step 3 and go to Step 4, if ready to do the O.D. measurement. Go to Step 3, if the O. D. measurement should be done later. d) No absorbance change up to 48 hours of storage. up to 48 hour storage is possible General Protocol at a Glance Read Technical Information carefully prior to using this General Protocol - 5 - Cell Counting Kit 8 CK04