The study of cell proliferation and cell viability requires the accurate quantification of the number of viable cells in a cell culture. Therefore, assays for calculating cell viability are necessary for optimizing cell culture conditions, evaluating cell growth factors and nutrients, discovering novel antibiotics and anti-cancer drugs, evaluating toxic effects of environmental pollutants and cell mediated toxicity and studying programmed cell death (apoptosis).
The Cell Viability assay kit provides a convenient, sensitive, quantitative and reliable assay for determining the number of viable cells in a given culture. This homogeneous Colorimetric Assay: is based on the conversion of a tetrazolium salt MTT, a pale yellow substrate, to formazan, a purple dye. This cellular reduction reaction involves the pyridine nucleotide cofactors NADH/NADPH and is only catalyzed by living cells. The formazan product has a low aqueous solubility and is present as purple crystals. Dissolving the resulting formazan with a solubilization buffer permits the convenient quantification of product formation. The intensity of the product color, measured at 550-620nm, is directly proportional to the number of living cells in the culture. Reagents in the kit have been carefully formulated and optimized for sensitivity, assay robustness and automation.
Sensitive and accurate. As low as 950 cells can be accurately quantified
Fast. High-throughput assay using 96-well plates allows simultaneous processing tens of thousands of samples per day
Homogeneous and convenient. "Mix-incubate-measure" type assay. No wash and reagent transfer steps are involved
Robust and amenable to HTS. Z’ factors of 0.5 and above are observed
Can be readily automated with HTS liquid handling systems.
Applications:Cell Proliferation: effects of cytokines, growth factor, nutrients
Cytotoxicity and Apoptosis: evaluation of toxic compounds, anti-cancer antibodies, toxins, environmental pollutants etc
Drug Discovery: high-throughput screen for toxic and anticancer drugs.
Kit Contents:Assay BufferSolubilization Solution
Note: Control Reagent: Saponin not supplied with kit
Storage Conditions:Store the Reagent at -20°C. Keep Assay Buffer and Solubilization Solution at 4 °C and room temperature, respectively. Shelf life: 12 month after receipt.
Precautions:Reagents are for research use only. Normal precautions for laboratory reagents should be exercised while using the reagents. Please refer to Material Safety Data Sheet for detailed information.
Procedures:The Cell Viability assay is based on the conversion of MTT to purple formazan by metabolically active cells. For most cells, this reducing reaction takes 3 to 5 hours. The produced formazan is solubilized and the resulting colored solution is quantified with a microplate reader. Although most culture media contain phenol red, phenol red does not interfere with the assay. All data in Technical Notes: were obtained in culture media containing phenol red.
Procedure using 96-well plate:1. Plate and culture cells (80ul per well) in a clear bottom 96-well tissue culture plates. Typical culture medium contains DMEM, 10% fetal bovine serum, antibiotics (penicillin/streptomycin, gentamycin, etc), amino acids and other nutrients. Assays can be performed on either adherent cells or cells in suspension. The number of cells can vary from 1000 to 80,000 per well. The volume can vary from 50 to 150ul, although 80ul is used in this example. In addition to the test samples, one must include control wells of culture medium containing no cells or cells treated with a toxic reagent such as 0.1% saponin
2. Add test compounds and controls and incubate cells for the desired period of time (typically overnight). It is recommended that assays be run in duplicate or triplicate. A volume of 20ul in phosphate buffered saline (PBS) or culture medium is recommended for the test compounds and controls. The Control reagent can be conveniently reconstituted with 5ml PBS (1% saponin)
3. Reconstitute the Reagent with Assay Buffer. First equilibrate the Reagent and Assay Buffer to room temperature. Then simply combine Assay Buffer and the Reagent by pipetting a small volume (e.g. 1ml) buffer to the Reagent tube. Vortex briefly and pipet the reconstituted solution to the Assay Buffer bottle. Repeat this step to transfer all Reagent to the Assay Buffer bottle. Mix by inversion until the Reagent is thoroughly dissolved. After this is done, mark the bottle label as Reconstituted Reagent. Note: Fresh reconstitution is recommended although the reconstituted reagent is stable for up to 4 weeks when stored at -20 °C
4. Add 15ul (per 80ul cell culture) of reagent per well and incubate for 4 hours at 37°C. The volume of the reagent should be adjusted depending on the volume of cell culture
5. Add 100ul of the Solubilization Solution. Mix gently on an orbital shaker for one hour at room temperature. The volume of the Solubilization Solution should be adjusted depending on the volume of cell culture. If precipitation occurs in the Solubilization Solution, place the bottle in a warm water bath or at 37°C and shake to dissolve precipitates
6. Measure OD570nm for each well on an absorbance plate reader. Maximum absorbance of the formazan dye lies between 560 and 590nm. If desired, the OD measurement can be performed the following day. In this case, it is recommended to seal the plate to minimize evaporation.
Data Analysis:Determine the average of the blank controls and subtract this amount from all absorbance values. Plot the corrected absorbance values at 570nm against the concentration of the test compound. Determine the EC50 value for cell proliferation and IC50 value for cytotoxic compound by non-linear regression analysis using Prism or any other Data Analysis: tools.
Technical Notes:The Cell Viability assay kit provides a convenient and homogeneous assay for cell proliferation and cytotoxicity in multi-well plates. Reagents of the kit have been carefully formulated and optimized for sensitivity, assay robustness and automation. Key Features: of the kits include:Safe. Non-radioactive assay (cf. 3H-thymidine incorporation assay)
Sensitive and accurate. As low as 950 cells can be accurately quantified
Fast. High-throughput assay using 96-well plates allows simultaneous processing tens of thousands of samples per day
Homogeneous and convenient. "Mix-incubate-measure" type assay. No wash and reagent transfer steps are involved
Robust and amenable to HTS: Z’ factors of 0.5 and above are observed
Can be readily automated with HTS liquid handling systems.
