Clearly Distinguishes Living Cells From Dead Cells And Debris

December 27, 2016

Duke University
Immunology
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Company:

Thermo Fisher Molecular Probes

Product Name:

LIVE/DEAD Fixable Far Red Dead Cell Stain Kit, for 633 or 635 nm excitation

Catalog Number:

L34973

The goal of this research was to clearly identify living splenocytes 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. 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

Must use PBS instead of FACS buffer to dilute LIVE/DEAD, because the proteins present in FACS buffer will bind the LIVE/DEAD and prevent dead cells binding LIVE/DEAD.

Results Summary

In the absence of LIVE/DEAD APC (left plot), all cells appear to be very negative for LIVE/DEAD APC. With the addition of LIVE/DEAD APC, dead cells were clearly distinguished from live cells (right plot), with live cells showing low/negative expression for LIVE/DEAD APC.

DOI or PMID #

N/A

Additional Notes

N/A

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Summary

The Good

Clearly distinguishes healthy, living cells from 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

Dead cell exclusion is a necessary first step in any flow cytometry-based experiment, and this reagent makes dead cell exclusion simple and robust.

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