Cell cytotoxicity / Proliferation assay cell type - adipose stem cells

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 2 matching solutions for this experiment

MTT Assay solution

Sigma-Aldrich

Upstream tips	Protocol tips	Downstream tips
	After 24 hours, the culture medium was aspirated and the cells were rinsed twice with PBS (Roche). The MTT solution was prepared by adding 8 mg of MTT to 10 mL fresh DMEM and 500 μL of MTT solution was added to each well. Four hours later, the MTT solution was removed and each well was incubated with 500 μL isopropanol for 30 min.	The extinction values of the isopropanol-diluted formazan were measured at 570 nm wavelength. The viability index was determined by calculating the ratio of the extinction values of labeled cells compared to unlabeled control cells.

Protocol tips
After 24 hours, the culture medium was aspirated and the cells were rinsed twice with PBS (Roche). The MTT solution was prepared by adding 8 mg of MTT to 10 mL fresh DMEM and 500 μL of MTT solution was added to each well. Four hours later, the MTT solution was removed and each well was incubated with 500 μL isopropanol for 30 min.
Downstream tips
The extinction values of the isopropanol-diluted formazan were measured at 570 nm wavelength. The viability index was determined by calculating the ratio of the extinction values of labeled cells compared to unlabeled control cells.

Manufacturer protocol Publication protocol 1 Relevant paper

ATP Assay

Abcam

Upstream tips	Protocol tips	Downstream tips
	The amount of adenosine triphosphate (ATP) produced by investigated cells was performed using ATP assay kit (Abcam). All procedures were performed according to manufacturer's protocols.

Protocol tips
The amount of adenosine triphosphate (ATP) produced by investigated cells was performed using ATP assay kit (Abcam). All procedures were performed according to manufacturer's protocols.

Manufacturer protocol Publication protocol 1 Relevant paper

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