Principle

Comparison of Assay Procedure with MTT and Cell Count Reagent SF

Absorption Spectrum

Absorption spectrum of WST-8 formazan

Correlation with [3H]-Thymidine

Application for Cell Proliferation Assay

1. Prepare a cell suspension using an appropriate culture medium, count cells to the desired number and dispense 100 µl of suspension of cells in logarithmic growth phase into each well of a 96-well plate*1.
2. Pre-incubate the medium in CO2 incubator.
3. Add 10 µl of Cell Count Reagent SF to each well of the plate*2.
4. Incubate the medium for 1-4 hours in the incubator*3.
5. Measure the absorbance at 450 nm (calibration wavelength: 600 nm or higher) by micro plate reader*4.
   *1 Usable with media containing phenol red.
   *2 Sterilize the solution by filtration with 0.22 µm membrane, as necessary.
   *3 Consider the condition. The absorbance is different among the type or number of cells. How to stop the reaction:
   1) Cool down the plate to 4°C.
   2) Add 10 µl of 0.1mol/l HCl.
   3) Add 10 µl of 1w/v% SDS. Measure the absorbance within 24 hours.
   *4 Use 430-490 nm filter. Wavelength of maximum absorbance for formazan is around 460 nm.

Media

HeLa: DMEM1640 (10% FBS)
HL-60: RPMI1640 (10% FBS)

Incubation

Hela cells: 5% CO2, 37°C, 1 hour (■), 2 hours (▲)
HL-60: 5% CO2, 37°C, 1 hour (■), 3 hours (●)

Measurement Wavelength

450 nm (calibration wavelength: 650 nm)

Cytotoxicity Assay

1. Prepare a suspension of cells in logarithmic growth phase with 5,000 cells/well using an appropriate culture medium. Add 100 µl of the cell suspension to each well of a 96-well plate.
2. Pre-incubate the medium in CO2 incubator for 24 hours.
3. Add 10 µl of the prepared toxicant on each well of the plate.
4. Incubate the medium for 48 hours in CO2 incubator.
5. Add 10 µl of Cell Count Reagent SF to each well of the plate.
6. Incubate the medium for 1-4 hours in CO2 incubator.
7. Measure the absorbance at 450 nm (calibration wavelength: 600 nm or higher) by microplate reader.

1. M. Ishiyama, Y. Miyazono, K. Sasamoto, Y. Ohkura, K. Ueno, Talanta, 44, 1299 (1997)
2. H. Tominaga, M.Ishiyama, F. Ohseto, K. Sasamoto, T. Hamamoto, K. Suzuki and M. Watanabe, Anal. Commun, 36 (2), 47 (1999)

Cell Count Reagent SF (PDF 1.6MB)