Viability/Cytotoxicity Multiplex Assay KitCK17 Product Manual

A method for measuring the number of viable cells or that of dead cells is widely used as a cytotoxicity evaluation. In order to determine the cytotoxicity comprehensively, a toxicity evaluation by combining a plurality of evaluation methods has been increasingly demanded. Viability/Cytotoxicity Multiplex Assay Kit can be performed as a comprehensive cytotoxicity assay. Viability/Cytotoxicity Multiplex Assay Kit includes Cell Counting Kit-8 (CCK-8) and Cytotoxicity LDH Assay Kit-WST (LDH Assay Kit), which measures the number of viable cells and dead cells, respectively.
CCK-8 can determine the number of viable cells by measuring dehydrogenases activity in living cells and LDH Assay Kit can determine the number of dead cells by measuring the amount of released LDH from damaged cell membranes. LDH Assay Kit enables both homogeneous assay by adding the reagent solution in the kit to a cell culture medium directly and non-homogeneous assay by adding the reagent solution to a cell culture supernatant. Two index cytotoxicity evaluations with same cultured cells can be carried out by combining CCK-8 and the non-homogeneous assay in LDH Assay Kit.

	Code	Unit size	Components
Cell Counting Kit-8	CK04	500 tests	Cell Counting Kit-8	5 ml × 1
Cytotoxicity LDH Assay Kit	CK12	500 tests	Dye Mixture	× 1
			Assay Buffer	55 ml × 1
			Lysis Buffer	5.5 ml × 1
			Stop Solution	27.5 ml × 1

Store at 0-5℃

• Microplate reader with 450 nm and 490 nm filter
• CO₂ incubator
• 20, 100-200 μl 8-channel pipette
• 96-well tissue culture plate (flat-bottomed)
  • Round or V-bottomed plate for suspension cells in non-homogeneous assay.
• 96-well optically clear plate (flat-bottomed)
  • For non-homogeneous assay in LDH measurement.

The optimum conditions depend on types of cells. We recommend carrying out a preliminary experiment to optimize the concentration of cells and the incubation time.

CCK-8 assay for viable cell detection

Optimization of cell concentration and incubation time Cell viability assay

 

LDH Assay for cell death detection

・Homogeneous assay  Optimization of cell concentration Cytotoxicity assay

・Non-homogeneous assay Optimization of cell concentration Cytotoxicity assay

 

Experimental example for CCK-8 and LDH assay in same cells

• Cell viability and cytotoxicity assay

 

　Serial dilution procedure　

The ideal number of cells and the incubation time for color development should be determined by a preliminary experiment. Prepare cells without addition of test substances in culture medium.

Optimization of Cell Concentration and Incubation Time

1. Collect cells and wash them with the medium. Prepare 5×10⁵ cells/ml cell suspension in the medium.
2. Prepare 100 μl of 2-fold serially diluted cell suspension in a 96-well tissue culture plate.
   • Refer the Serial Dilution Procedure.
3. Incubate cell suspension at 37°C for an appropriate time in a CO₂ incubator.
4. 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 the O.D. reading.
5. Incubate the plate at 37°C for 1 - 4 hours in the CO₂ incubator.
   • Color intensity depends on types of cells. Measure the absorbance every hour and find the optimum incubation time which allows absorbance to be in following range.
     for cytotoxicity assay ： 0.5 - 1.5
     for proliferation assay ： 0.3 - 0.8
6. Measure the absorbance at 450 - 490 nm by using a microplate reader.

Experimental example ： the number of cells vs. absorbance

 

 

Cell Viability Assay

1. Collect cells and wash them with the medium. Prepare the cell suspension at the optimum concentration which is determined in a preliminary experiment.
2. Add 100 μl of the cell supension to each well of a 96-well tissue culture plate.
   • For adherent cells, please incubate the culture plate overnight to attach the cells to the plate.
3. Add 10 μl of the medium containing test substance that adjusted to the desired concentration. Incubate the plate at 37°C for an appropriate time(e.g. 6, 12, 24, or 48 hours) in a CO₂ incubator.
   • In case the test substance has color or reducing activity, prepare “Blank” well which includes test substance, CCK-8, and medium without cells.
4. Add 10 μl of the 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.
5. Incubate the plate at 37℃ for 1 - 4 hours in the CO₂ incubator.
   • Please refer to the determined optimum incubation time in preliminary experiment.
6. Measure the absorbance at 450 - 490 nm by using a microplate reader.

 

Calculation of Cell Survival Rate

Calculate the average absorbance from each triplicate set of wells and subtract the absorbance of “Blank” from each absorbance of “Sample” and “Control” in the following equation.

There are two methods: homogeneous assay and non-homogeneous assay, please choose the assay method according to your experiments.

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Preparation of Reagent

1. To dissolve the powder, add 5 ml of Assay Buffer to the Dye Mixture vial. Close the cap and dissolve the contents completely.
2. Add the whole volume of the mixture prepared in 1) to the Assay Buffer bottle.
   • Store the Working Solution at 0-5℃ and protect it from light. It is stable for 6 months.

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Homogeneous assay

Optimization of Cell Concentration

1. Collect cells and wash them with the medium. Prepare 5×10⁵ cells/ml cell suspension in the medium.
2. Prepare 100 μl of 2-fold serially diluted cell suspension in a 96-well tissue culture plate.
   • Refer the Serial Dilution Procedure.
3. Incubate cell suspension at 37℃ for an appropriate time in a CO₂ incubator.
4. Add 10 μl of Lysis Buffer to each well of the high control.
5. Incubate the plate at 37℃ for 30 minutes in the CO₂ incubator.
6. Add 100 μl of Working Solution to each well. Protect the plate from light and incubate it at room temperature for 30 minutes.
7. Add 50 μl of Stop Solution to each well.
8. Measure the absorbance at 490 nm by using a microplate reader.
   • Plot the data by setting cell concentrations on the x-axis and absorbances on the y-axis. The plot should have two linear lines, one representing High Control and another Low Control. Determine the optimum concentration of cells based on the followings.
     Eliminate the concentration of cells that has absorbances higher than 2.0.
     Choose the concentration of cells that has at least 0.2 difference in the absorbance between high control and low control.

 

Cytotoxicity Assay

1. Add 50 μl of cell suspension to each well of a flat-bottom 96-well tissue culture plate.
   • Prepare the double concentration of cell suspension which is confirmed in the preliminary experiment.
     For adherent cells: incubate the plate at 37℃ overnight in a CO₂ incubator to allow the cells to adhere and then replace the medium with 50 μl of fresh medium.
2. Add 50 μl of medium containing test substance that adjusted to the desired concentration (Refer to Table 1).
3. Incubate the plate at 37℃ for an appropriate time in a CO₂ incubator.
4. Add 10 μl of Lysis Buffer to each well of the high control. Incubate the plate at 37℃ for 30 minutes in a CO₂ incubator.
5. Add 100 μl of Working Solution to each well. Protect the plate from light and incubate it at room temperature for 30 minutes.
6. Add 50 μl of Stop Solution to each well.
7. Measure the absorbance at 490 nm by using a microplate reader.

 

Calculation of Cytotoxicity

Calculate the average absorbance from each triplicate set of wells and subtract the absorbance of background from each absorbance of test substance, high control, and low control. Determine the percent cytotoxicity by the following equation.

 

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Non-homogeneous assay

Optimization of Cell Concentration

1. Collect cells and wash them with the medium. Prepare 5×10⁵ cells/ml cell suspension in the medium.
2. Prepare 2-fold serially diluted cell suspension in a 96-well tissue culture plate.
   • Refer the Serial Dilution Procedure.
3. Add 100 μl of the cell suspension and 100 μl of the medium to each well.
4. Incubate the plate at 37℃ for an appropriate time in a CO₂ incubator.
5. Add 20 μl of Lysis Buffer to each well of the high control.
6. Incubate the plate at 37℃ for 30 minutes in the CO₂ incubator.
7. Centrifuge the plate at 250 × g for 2 minutes to precipitate the cells (for suspension cells).
8. Transfer 100 μl of the supernatant from each well to a 96-well clear plate.
9. Add 100 μl of Working Solution to each well. Protect the plate from light and incubate it at the room temperature for 30 minutes.
10. Add 50 μl of Stop Solution to each well.
11. Measure the absorbance at 490 nm by using a microplate reader.
    • Plot the data by setting concentration of cells on the x-axis and absorbances on the y-axis. The plot should have two linear lines, one representing High Control and another Low Control. Determine the optimum concentration of cells based on the followings.
      Eliminate the concentration of cells that has absorbances higher than 2.0.
      Choose the concentrations of cells that has at least 0.2 difference in the absorbance between high control and low control.

 

Cytotoxicity Assay

1. Add 100 μl of the cell suspension to each well of a 96-well tissue culture plate.
   • For adherent cells: incubate the plate at 37℃ overnight in a CO₂ incubator to allow the cells to adhere and then replace the medium with 100 μl of fresh medium.
2. Add 100 μl of the medium containing test substance that adjusted to the desired concentration (Refer to Table 2).
3. Incubate the plate at 37℃ for an appropriate time in a CO₂ incubator.
4. Add 20 μl of Lysis Buffer to each well of the high control. Incubate the plate at 37℃ for 30 minutes in the CO₂ incubator.
5. Centrifuge the plate at 250 × g for 2 minutes to precipitate the cells (for suspension cells).
6. Transfer 100 μl of the supernatant from each well to each well of a new 96-well clear plate.
7. Add 100 μl of the Working Solution to each well. Protect the plate from light and incubate it at room temperature for 30 minutes.
8. Add 50 μl of Stop Solution to each well.
9. Measure the absorbance at 490 nm by using a microplate reader.
    

Calculation of Cytotoxicity

Calculate the average absorbance from each triplicate set of wells and subtract the absorbance of background from each absorbance of test substance, high control, and low control. Determine the percent cytotoxicity by the following equation.
 

 

Cell Viability and Cytotoxicity Assay

1. Add 5000 cells/100 μl of HeLa cell to each well of a 96-well tissue culture plate.
2. Incubate the plate at 37℃ overnight in a CO₂ incubator.
3. Add 100 μl of the medium containing test substance that adjusted to the desired concentration.
4. Incubate the plate at 37℃ for one hour in the CO₂ incubator.
   • Settle the suitable incubation time for the experiment.
5. Add 20 μl of Lysis Buffer to each well of the high control at 30 minutes before the end of incubation time.

 

LDH assay using supernatant (under the procedure of Non-homogeneous assay)

1. Transfer 100 μl of the supernatant to a new 96-well clear plate.
2. Add 100 μl of the Working Solution to each well.
3. Protect the plate from light and incubate it at room temperature for 30 minutes.
4. Add 50 μl of Stop Solution to each well.
5. Measure the absorbance at 490 nm by using a microplate reader

 

CCK-8 assay using cultured medium with cells

1. Add 10 μl of CCK-8 to each well where cells have been cultured.
2. Incubate the plate at 37℃ for 3 hours in the CO₂ incubator.
   • Settle the suitable colorimetric reaction time for the experiment.
3. Measure the absorbance at 450 nm by using a microplate reader.

 Result of experimental example

Add 100 μl of medium to each well of a 96-well tissue culture plate. Then, add the cell suspension (5 x 10⁵ cells/ml) to the first well and mix by pipetting. This well contains the maximum number of cells (2.5 x 10⁴ cells/well). Transfer 100 μl from the first well to the next well, and mix by pipetting. Repeat this procedure.

Procedure for serial dilution