Cell cytotoxicity / Proliferation assay cell type - L-02

Cell cytotoxicity assays measure the ability of certain compounds or chemical mediators to reduce the viability of the cells. The term cell cytotoxicity assay can sometimes be used interchangeably with cell proliferation assay. Healthy living cells can be identified by the use of formazan dyes, protease biomarkers or by measuring ATP content. The formazan dyes are chromogenic products formed by the reduction of tetrazolium salts by dehydrogenases, such as lactate dehydrogenase (LDH) and reductases that are released during cell death. Common tetrazolium salts include INT, MTT, MTS and XTT. Cell cytotoxicity can also be measured by using the SRB and WST-1 assays. These assays can usually be used in a high-throughput fashion and can be quantitated by measuring absorbance, colorimetry or luminescence. All these assays require similar numbers of cell plating at the initiation, a time course of treatment with the cytotoxic agent and at least triplicates for each condition at every point of analysis. Cell shrinkage, plasma membrane blebbing, cell detachment, externalization of phosphatidylserine, nuclear condensation and ultimately DNA fragmentation are well-described features of apoptosis. The assays that rely on cell membrane integrity for their function, may not be able to quantify early apoptosis. Therefore, in order to distinguish early apoptotic vs. late apoptotic or necrotic cells, additional flow cytometry techniques can be used. A combination of Annexin V and PI (propidium iodide) can be used to distinguish early (Annexin V+/PI-) and late apoptotic (Annexin V+/PI+) cells. Sometimes, caspase assays are used in order to differentiate the stages of apoptosis.

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Found 3 matching solutions for this experiment

Upstream tips
- Completely thaw the 20ml size One Solution Reagent approximately 90 minutes at room temperature, or 10 minutes in a water bath at 37°C.
Protocol tips
- Cells were incubated with 20 μl of MTS for 3 h
Downstream tips
- To measure the absorbance later, add 25µl of 10% SDS to each well to stop the reaction. Store SDS-treated plates protected from light in a humidified chamber at room temperature for up to 18 hours.
Downstream tips
- If large variation of absorbance is present, please check for presence of bubble and break them; Mix reagents properly with accurate volumes as suggested.

- Incase of High background values, use the serum-free medium or the medium contains the serum less than 5%.
Upstream tips
- 3,000/well were seeded in a 96 well plate.

- Cells were pretreated with siRNA before performing an assay
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