CCK-8 Kit (Cell Proliferation and Toxicity Detection)

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

Instructions for Use:
1、 Create a standard curve (when measuring the specific number of cells)
1. First, count the number of cells in the prepared cell suspension using a cell counting plate, and then inoculate the cells.
2. Dilute the culture medium proportionally to form a cell concentration gradient. Generally, 3-5 cell concentration gradients are required, with 3-6 replicates per group.
3. After inoculation, culture the cells until they adhere to the wall, then add CCK-8 reagent to culture for a certain period of time and measure the OD value. Create a standard curve with cell number as the x-axis and OD value as the y-axis. According to this standard curve, the number of cells in unknown samples can be determined (the premise of using this standard curve is that the experimental conditions must be consistent to facilitate the determination of the number of cells inoculated and the culture time after adding CCK-8). ）

2、 Cell viability testing

1. Inoculate cell suspension (100 µ L/well) into a 96 well plate. Place the culture plate in the incubator for pre cultivation (at 37 ℃, 5% CO2).

2. Add 10 µ L of CCK-8 solution to each well (be careful not to generate bubbles in the well, as they can affect the OD reading).

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

4. Measure the absorbance at 450nm using an enzyme-linked immunosorbent assay (ELISA) reader.

5. If the OD value is not measured temporarily and plans to be measured in the future, 10 µ L of 0.1M HCl or 1% SDS (W/V) solution can be added to each well, and the culture plate can be covered and stored at room temperature in the dark. The absorbance will not change within 24 hours.

3、 Cell proliferation toxicity detection

1. Prepare 100 µ L of cell suspension in a 96 well plate. Pre culture the culture plate in the incubator for 24 hours (at 37 ℃, 5% CO2).

2. Add 10 µ L of the test substance at different concentrations to the culture plate. Incubate in the incubator for an appropriate period of time (e.g. 6, 12, 24, or 48 hours).

3. Add 10 µ L of CCK-8 solution to each well (be careful not to generate bubbles in the well, as they can affect the OD reading). If the substance to be tested has oxidizing or reducing properties, fresh culture medium can be replaced before adding CCK-8 (remove the culture medium, wash the cells twice with the culture medium, and then add new culture medium) to remove drug effects.

4. Incubate the culture plate in the incubator for 1-4 hours.

5. Measure the absorbance at 450 nm using an enzyme-linked immunosorbent assay (ELISA) reader.

6. If the OD value is not measured temporarily and plans to be measured in the future, 10 µ L of 0.1M HCl or 1% SDS (W/V) solution can be added to each well, and the culture plate can be covered and stored at room temperature in the dark. The absorbance will not change within 24 hours.

Vitality Calculation:

Cell viability (%)=[A (dosing) - A (blank)]/[A (0 dosing) - A (blank)] × 100

A (Medication): Absorbance of pores containing cells, CCK-8 solution, and drug solution

A (blank): Absorbance of wells with culture medium and CCK-8 solution without cells

A (0 dosing): absorbance of wells with cells and CCK-8 solution but without drug solution

Cell viability: Cell proliferation viability or cytotoxic viability

Notes:

① Suggest making a few wells first to explore the number of cells to be inoculated and the cultivation time after adding CCK-8 reagent.

② White blood cells may need to be cultured for a longer period of time.

③ When using a standard 96 well plate, the minimum inoculation amount of adherent cells should be at least 1000 cells per well (100 µ L culture medium). The sensitivity of detecting white blood cells is relatively low, so it is recommended to inoculate no less than 2500 cells per well (100 µ L culture medium). If you want to use a 24 well or 6-well plate for the experiment, please first calculate the corresponding inoculation amount for each well, and add CCK-8 solution at 10% of the total volume of the culture medium for each well.

④ If there is no 450nm filter, a filter with absorbance between 430-490nm can be used, but 450nm has the highest detection sensitivity.

⑤ The absorbance of phenol red in the culture medium can be eliminated by subtracting the absorbance of the background in the blank well during calculation, so it will not affect the detection.