Live / Dead assay mammalian cells - rat brain microvascular endothelial cells

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
supplemented with 15% fetal bovine serum (FBS) (Biochrom AG), 1 ng/ml basic fibroblast growth factor (Roche Applied Sciences), 100 µg/ml heparin (Biochrom AG), 5 µg/ml gentamycin and 4 µg/ml puromycin [81] (medium I) at 37°C with a humidified atmosphere of 5% CO2, for 2 days.
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