Flow Cytometry Viability Stain

November 14, 2017

University of Colorado Denver
Medicine
Professional Research Assistant

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Company:

Thermo Fisher Scientific

Product Name:

eBioscience™ Fixable Viability Dye eFluor™ 506

Catalog Number:

65-0866-14

Our research is aimed at understanding BAL cell mortality in response to different lung inhalants/particulates. In these experiments, we treated mice in vivo with different inhalants/particulates and used this FVD eFluor 506 to determine BAL cell viability as a part of a larger flow cytometric panel. We wanted to quantify the effects of various treatments on cell death/membrane permeability as compared to controls under different conditions. We needed a fixable viability dye so that we could FACS fix our cells, and we selected this product because we use many other eBioscience products, and the product was reasonably priced. It also comes in a variety of flurophores - this eFluor 506 detected under Amcyan on our machine and worked nicely with the rest of our BAL panel.

Experimental Design and Results Summary

Application

Flow Cytometry

Starting Material

BAL cells

Protocol Overview

Cells can be stained with FVD separately or as a part of an antibody panel master mix. The FVD should be equilibrated to room temperature prior to staining, and should spend as little time as possible diluted in an antibody mix or diluent prior to staining. We stained our cells at 4C for 30 mins (on an ice bucket covered with foil or in a refrigerator). Cells can then be washed with azide-free FACS buffer and stained or fixed.

Tips

Diluent FVD right before applying to cells. We kept our product stored at -80 in 5-10ul aliquots at all times when not using it. We kept it wrapped in foil while it was equilibrating to room temperature. We found this eliminated a lot of variability in our resultant staining.

Results Summary

This FVD gives you two distinct populations of live vs dead (membrane impermeable vs membrane permeable) cells. It is easy to determine cell viability based on fluorescence intensity in the flow cytometry data.

DOI or PMID #

N/A

Additional Notes

N/A

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Summary

The Good

Easy to tell live vs dead cells.

The Bad

Product does not tolerate sitting in an antibody master mix for very long without losing staining intensity/population clarity.

The Bottom Line

A good fixable viability dye. Stains clear populations of live & dead cells. Reliable staining, if manufacturer protocol is followed. Comes in a variety of fluorphores which makes it easy to work into existing antibody panels. Overall, a very good product.

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