Cell cytotoxicity / Proliferation assay cell type - HeLa

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 5 matching solutions for this experiment

Upstream tips
- Pre-incubate the plate for 24 hours in a humidified incubator. Be careful not to introduce bubbles to the wells, since they interfere with the O.D. reading.
Downstream tips
- After cells were washed they were irradiated with 5 J/cm2 light dose using a 635 nm laser at a power density of 20 mW/cm2.
Protocol tips
- 100 ul Fixative/Denaturing solution is added and incubate for 30 minutes at room temperature. 100 μl of Substarte solution is added to each well and the color reaction is stopped after 10 minutes with 25 μl 1 M H2SO4.
Downstream tips
- Incase of high background reduce cell concentartion.
Protocol tips
- 250 μg/ml of MTT is added and incubated for 4 extra hours
Downstream tips
- Absorbance is measured at a wavelength of 570 nm with background subtraction at 630-690 nm.
Protocol tips
Add 10 μl of the MTT
labeling reagent (final concentration 0.5 mg/ml)
and incubate the microplate for 4 h in a humidified atmosphere (e.g., +37°C, 5 - 6.5% CO2 ).
Downstream tips
- LDH activity was measured after 24 h of incubation
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