Cell Counting Kit-8, abbreviated as CCK-8 kit, is an alternative to MTT method. It is a rapid and highly sensitive detection kit based on WST (water-soluble tetrazolium salt, chemical name: 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium). It can be used for drug screening, cell proliferation test, cytotoxicity test, tumor drug sensitivity test and so on.

Instructions for use:

First. make a standard curve (when determining the specific number of cells)

1. The number of cells in the prepared cell suspension was counted with a cell counting board, and then the cells were inoculated;

2. According to the proportion, the medium was diluted into a cell concentration gradient. Generally, 3-5 cell concentration gradients were made, with 3-6 multiple holes in each group;

3. After inoculation, the cells were cultured to adhere to the wall, and then cultured with CCK-8 reagent for a certain period of time, the OD value was measured, and a standard curve with the number of cells as abscissa (X axis) and OD value as ordinate (Y axis) was made. The number of cells in unknown samples can be determined according to this standard curve (the premise of trying this standard curve is that the experimental conditions are consistent, which is convenient to determine the number of cells inoculated and the culture time after adding CCK-8.

Second. cell activity test

1. Inoculate cell suspension (100 μL/well) in 96-well plate. The culture plate was pre-cultured in the incubator (at 37℃, 5% CO2);

2. Add 10 μL CCK-8 solution to each hole (be careful not to form bubbles in the hole, which will affect the reading of the OD value);

3. Incubate the culture board in the incubator for 1-4 hours;

4. The absorbance at 450 nm was determined by enzyme labeling instrument;

5. If the OD value is not determined for the time being and is going to be measured later, 10 μL 0.1M HCl or 1%SDS (W/V) solution can be added to each hole, and the culture plate can be shielded from light and stored at room temperature. The absorbance does not change within 24 hours.

Third. Cell proliferation-toxicity test

1. 100 μL cell suspension was arranged in 96-well plate. The culture plate was pre-cultured in the incubator for 24 hours (at 37℃, 5%CO2);

2. Add 10 μL of different concentrations of substances to the culture plate. Incubate in the incubator for an appropriate period of time (for example, 6, 12, 24 or 48 hours);

3. Add 10 μL CCK-8 solution to each hole (be careful not to form bubbles in the hole, which will affect the reading of the OD value). If the substance to be tested is oxidizing or reductive, the fresh medium can be replaced before adding CCK-8 (remove the medium, wash the cells with the medium twice, and then add a new medium) to remove the drug effect;

4. Incubate the culture board in the incubator for 1-4 hours;

5. The absorbance at 450 nm was determined by enzyme labeling instrument;

6. If the OD value is not determined for the time being and is going to be measured later, 10 μL 0.1M HCl or 1%SDS (W/V) solution can be added to each hole, and the culture plate can be shielded from light and stored at room temperature. The absorbance will not change within 24 hours.

Vitality calculation: Cell viability (%) = [A (drug addition)-A (blank)] / [A (0 drug addition)-A (blank)] × 100

A (drug addition): absorbance of pores with cells, CCK-8 solutions and drug solutions

A (blank): absorbance of pores with medium and CCK-8 solution without cells

A (0 drug addition): absorbance of holes with cells, CCK-8 solution but no drug solution

Cell viability: cell proliferation or cytotoxicity

Note:

1. It is recommended to do a few holes to find out the number of inoculated cells and the culture time after adding CCK-8 reagent.

2. Leukocytes may need to be cultured for a long time.

3. When using standard 96-well plate, the minimum inoculation amount of adherent cells is at least 1000/well (100 μL medium). The sensitivity for the detection of leukocytes is relatively low, so it is recommended that the inoculation amount is not less than 2500/well (100 μL medium). If you want to use a 24-well plate or a 6-well plate, first calculate the corresponding amount of inoculation per well and add CCK-8 solution according to 10% of the total volume of the culture medium per well.

4. If there is no filter of 450 nm, the filter with absorbance between 430 and 490 nm can be used, but the sensitivity of 450 nm is the highest.

The absorbance of phenol red in the medium can be eliminated by deducting the background absorbance from the blank hole, so it will not affect the detection.

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