Live / Dead assay mammalian cells - 3T3-L1

An alternative to culture-based cell death detection is an assessment of other cell viability indicators using fluorescent dyes, including membrane potential and membrane integrity. Live/Dead assays differentiates live and dead cells using membrane integrity as a proxy for cell viability and are based on a fluorescent staining procedure followed by detection using flow cytometry. However, samples preparation for such flow cytometry-based techniques could be challenging. Cell harvesting by trypsinization, mechanical or enzymatic cell disaggregation from tissues, extensive centrifugation steps, may all lead to preferential loss of apoptotic cells. To overcome this strictly follow manufacturers instruction of the detection kit.

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Protocol tips
Add 100 uL 2uM Calcein AM in PBS to each well.

Incubate at 37°C for 1h .
Downstream tips
Serum in cell culture medium may contain esterase activity, which can increase background fluorescence. Cells can be rinsed in PBS at this step to reduce background caused by residual serum
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