Live / Dead assay mammalian cells - L29 mouse fibroblast

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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Found 4 matching solutions for this experiment

Upstream tips
Prepare a staining solution of 2 uM calcein AM/4 uM EthD-III by adding 5 uL of 4 mM calcein
AM and 20 uL of 2 mM EthD-III to 10 mL of PBS or other serum-free buffer or medium
Protocol tips
Wash cells before adding calcein AM/EthD-III staining
solution to cover the cell monolayer.

Incubate the cells for 30-45 minutes at room temperature.

Protocol tips
Add 4 μM of calcein-acetomethoxy (calcein AM-5 μL) and 2 mM of ethidium-bromide (20 μL).

Incubate this mixture for 30–45 min at room temperature in the dark
Protocol tips
Add 8 µM ethidium homodimer and 2 µM calcein acetoxymethyl in PBS and incubate for 30~45 min at room temperature
Protocol tips
Add 100 µl of assay solution to cells and incubate the mixture at 36.5 °C in 5 % CO2 atmosphere for 15 min.
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