Looking for Cell Counting Kit-8 protocol?

The Cell Counting Kit-8 is a colorimetric assay kit used to measure cell proliferation and cytotoxicity. It is a ready-to-use solution that can be added directly to the cell media for fast, high-throughput screening without a solubilization process obtaining highly reproducible and accurate results.

Cell Counting Kit-8 can be utilized to do cell number counting, cell prolifiration or cytotoxicity assay based on your requirments.  So it make a tiny protocol deferences when you apply different assay, while the majority of  operation protocol are the same. However, Some equipment and materials should be prepared before assay: 1). Plate reader (450 nm filter), 2). 96-well plate, 3). 10 µl, 100-200 µl and multi-channel pipettes.

 



More detailed manipulation procedures for Cell number counting and Cell proliferation and cytotoxicity sssays from Abbkine CCK-8 Kit are listed below for your references,

Cell Number Determination Protocol

1) Inoculate cell suspension (100 µl/well) in a 96-well plate. Also prepare wells that contain known numbers of viable cells (to create a calibration curve in step 5). Pre-incubate the plate in a humidified incubator (e.g., at 37 °C, 5% CO2).

2) Thaw the CCK-8 on the bench top or in a water bath at 37 °C if it is frozen. Note: It takes about 30 minutes on the bench top at 25 °C or 5 minutes in a water bath at 37 °C.

3) Add 10 µl of the CCK-8 solution to each well of the plate. Note: Be careful not to introduce bubbles to the wells, since they 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.

Note: 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.

Cell Proliferation and Cytotoxicity Assay Protocol

1) Dispense 100 µl of cell suspension (5000 cells/ well) in a 96-well plate.

2) Pre-incubate the plate for 24 hours in a humidified incubator (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 an appropriate length of time (e.g., 6, 12, 24 or 48 hours) in the incubator.

5) Thaw the CCK-8 on the bench top or in a water bath at 37 °C if it is frozen. Note: 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 they 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 microplate reader.

Note: 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.

Be attention to 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 Abbkine CCK-8 assay.