Live / Dead assay bacteria - Staphylococcus epidermidis

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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1 year ago

1 year ago by M. Daecher Germany

Live/dead assay Bacteria

Hello everyone! I am going to do a live/dead assay for my cells and I saw that I can use both fluorescence and absorbance as my detection method. Is there a difference in the results depending on the method? Is one method preferred over the other in certain situations?

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Protocol tips
Add 10 μL colouring reagent (comprising nine parts WST solution and one part electron mediator reagent for Gram-negative bacteria, or nine parts WST solution and one part eight-fold diluted electron mediator reagent for Gram-positive bacteria).

Incubate the plate in the incubator for 2–3 h at 35 °C
Protocol tips
Add a volume of BacTiter-Glo™ Reagent equal to the volume of cell culture medium present in each well.

Mix contents briefly on an orbital shaker and incubate for five minutes.
Protocol tips
Add 3 µL of the dye mixture for each mL of the bacterial
suspension.

Mix thoroughly and incubate at room temperature in the dark for 15 minutes
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