Removing Dead Cells From Flow Cytometry Analysis Using A Blue Live/Dead Stain Kit

March 03, 2017

Albert Einstein College of Medicine
Clinical Research
Pre-doctoral researcher

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Ease-of-Optimization

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Company:

Molecular Probes by Life Technologies (ThermoFisher Scientific)

Product Name:

LIVE/DEAD Fixable Blue Dead Cell Stain Kit

Catalog Number:

L23105

For most of our flow cytometry protocols we do not fix the cells prior to data acquisition, so we are able to use a simple DAPI stain to identify and remove dead cells from our analysis. When we do need to fix (i.e. for our intracellular staining protocols), however, we use the live/dead fixable blue dead cell staining kit by Life Technologies. We selected it based on its utility in multi-color panels and preserved function following the addition of a fixative reagent to each sample.

Experimental Design and Results Summary

Application

Flow cytometry

Starting Material

Human whole blood (100 ul)

Protocol Overview

Dissolve 1 vial of the stain with the DMSO included in the kit. After preparing the samples/cells (wash, centrifuge, etc.), add 1 ul of the blue L/D stain to each sample tube and incubate on ice for 30 minutes (no light). Then, perform a wash step and complete the remainder of your staining protocol followed by the required fixation (formaldehyde) steps.

Tips

Optimize the protocol (in completion) on controls or discarded samples prior to use.

Results Summary

Results are generated following flow cytometric analysis. The blue L/D stain should be excited by the UV channel with the detection filters 450/50 nm. Once clean separation of live and dead cell populations is obtained, the live cells can be gated on for further gating and analysis.

DOI or PMID #

N/A

Additional Notes

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Summary

The Good

Simple protocol, inexpensive, easy to use, reliable. Dye is separated into multiple vials, providing additional longevity from a single kit.

The Bad

Like all flow cytometry protocols, make sure to optimize the dilution prior to use on prescious samples.

The Bottom Line

I recommend this product to anyone who needs a reliable live/dead stain for flow cytometry protocols that involve fixing. We use this reagent in panels comprised of 7+ colors and have never experienced any problems with the product. Multiple other color options (other than blue) are available from the company to best fit the needs of different multi-color experiments.

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