General Information

Cell Counting Kit-8 (CCK-8) allows for very convenient assays using the highly water-soluble tetrazolium salt WST-8 [2- (2-methoxy-4-nitrophenyl) -3- (4-nitrophenyl) -5- (2,4-disulfophenyl) -2H, tetrazolium monosodium salt] (Patent No. WO97 / 38985) produces a water-soluble formazan dye on reduction in the presence of an electron carrier. Cell Counting Kit-8 is a one-bottle solution; no pre-mixing of components is required.

Cell Counting Kit – 8, non-radioactive, allows sensitive colourimetric assays for the determination of the number of viable cells in the cell proliferation and cytotoxicity assays. WST-8 is reduced by dehydrogenases in cells to give a yellow product (formazan), which is soluble in a tissue culture medium. The amount of 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, TXT, MTS or WST-1.

Advantages

• The solution from a bottle, ready to use
• No organic solvents or isotopes required
• No collection, washing and solubilization steps
• More sensitive than MTT, TXT, MTS or WST-1

Storage

CCK-8 is stable for 3 years at 2-8 ° C with protection against light. It can also be stored at -20 ° C. Repeated thawing and freezing causes a rise in the background, which interferes with the assay.

How To Use The Cellular Counting Kit-8

1. Equipment and materials needed

• plate reader (450 nm filter)
• 96-well plate
• 10 μl, 100-200 μl multichannel pipettes
• CO2 incubator

2. Protocol

• Cell proliferation assay

1) Inoculate the cell suspension (100 μl / well) into a 96-well plate. Also, prepare wells containing a known number of viable cells (to create a calibration curve in step 5). Pre-incubate the plate in a humidified container incubator (eg 37 ° C, 5% CO2).
2) Thaw CCK-8 on a benchtop or in a 37 ° C water bath if frozen.
Note: It takes about 30 minutes on the bench at 25 ° C or 5 minutes in a 37 ° C water bath.
3) Add 10 μl of CCK-8 solution to each well of the plate.
Note: Be careful not to introduce bubbles into the wells as they interfere with the outside diameter. 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 was obtained from wells containing known numbers of viable cells.
Note: To measure absorbance later, add 10 µl of 1% w / v SDS to each well, cover the plate, and store protection from light at room temperature. No change in absorbance should be observed for 48 hours.

• Cytotoxicity assay

1) Dispense 100 µl of cell suspension (5000 cells/well) into a 96-well plate.
2) Pre-incubate the plate for 24 hours in a humidified incubator (eg, 37 ° C, 5% CO2).
3) Add 10 µl of various concentrations of a toxicant to the plate culture medium.
4) Incubate the plate for a suitable period of time (eg 6, 12, 24 or 48 hours) in the incubator.
5) Thaw CCK-8 on a benchtop or in a 37 ° C water bath if frozen.
Note: It takes about 30 minutes on the workbench at 25 ° C or 5 minutes in the 37 ° C water bath.
6) Add 10 µl of CCK-8 solution to each well of the plate. Be careful not to introduce bubbles into the wells, as they interfere with the outside diameter reading.
7) Incubate the plate for 1-4 hours in the incubator. Measure the absorbance at 450 nm using a microplate reader.

Background Control

•  A slight spontaneous absorbance occurs around 460 nm in a culture medium incubated with CCK-8. This background absorbance depends on the culture medium, the pH, the incubation time and the duration of exposure to light.
• Typical background absorbance after 2 hours incubation is 0.1 to 0.2 absorbance units. To correct this, prepare one or more cell-free control wells and subtract the average absorbance of the control wells from that of the other wells.
• During a 5-hour experiment, the absorbance of the CCK-8 solution does not increase in the room.
  temperature.

Precautions

• Since the CCK-8 assay is based on the detection of dehydrogenase activity in viable cells, the conditions or chemicals that affect dehydrogenase activity in viable cells can cause discrepancies between the actual viable cell number and cell number determined using the CCK-8 assay.
• WST-8 can react with reducing agents to generate WST-8 formazan. Please check the bottom of whether reducing agents are used in cytotoxicity assays or cell proliferation assays.
• Be careful not to introduce bubbles into the wells as they interfere with the outside diameter. reading.
• Culture media containing phenol red can be used with this kit for cell viability assays.
• Membrane filtration is recommended for sterilization of the CCK-8 solution, if necessary.
• Incubation time varies depending on the type and number of cells in a well. Generally, leukocytes give faint staining, hence a long incubation time (up to 4 hours) or a large number of cells (~ 105 cells/well) may be required.
• Since the cytotoxicity of this kit is very low, further colour development is possible after reading.
  absorbance.