An Assay Kit to Exclude Dead Cells

December 22, 2016

Duke University
Immunology
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Company:

Thermo Fisher Molecular Probes

Product Name:

LIVE/DEAD Fixable Violet Dead Cell Stain Kit, for 405 nm excitation

Catalog Number:

L34955

The goal of this research was to clearly identify living human and mouse leukocytes for downstream phenotypic analysis by flow cytometry.

Experimental Design and Results Summary

Application

Flow Cytometry

Starting Material

Mouse Splenocytes

Protocol Overview

Prepare reagent as described by manufacturer. After obtaining single cell suspension and lysing red blood cells, resuspend cells in LIVE/DEAD diluted in PBS (1:5,000, 100 uL for one million cells) and incubate cells on ice in the dark for 20 minutes. Generally also include Fc block in LIVE/DEAD dilution. Wash cells once in FACS buffer (PBS with 0.1% FBS) after staining with LIVE/DEAD. Stain with isotype control or desired surface antibody (1:250-1000 dilution, 100 uL per million cells) for 20 minutes on ice.

Tips

Cannot use FACS buffer to stain cells with LIVE/DEAD, because LIVE/DEAD will be bound by proteins present in the FBS and will therefore not adequately stain dead cells.

Results Summary

In the absence of LIVE/DEAD (left plot, empty histogram in right plot), all cells appear to be very negative for LIVE/DEAD Violet. With the addition of LIVE/DEAD, dead cells were clearly labelled with LIVE/DEAD Violet (middle plot, gray histogram in right plot), while live cells were low/negative for LIVE/DEAD Violet.

DOI or PMID #

N/A

Additional Notes

N/A

Image Gallery

Summary

The Good

Very easy to use, clearly distinguishes dead cells.

The Bad

Nothing, really - it needs to be stained in PBS and not FACS buffer, which just adds a reagent.

The Bottom Line

This reagent is the standard first step in the lab's flow cytometry staining protocol.

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