Good Dye, Works to Stain Dead Cells

February 12, 2016

BlueskyReddit
University of California, San Diego
Department of Pediatrics
Postdoctoral Fellow

Overall

Quality of Results

Ease-of-Optimization

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Company:

Affymetrix eBioscience

Product Name:

Fixable Viability Dye eFluor® 506

Catalog Number:

65-0866-14

This dye is good; it took a few optimization tries, but the dye works nicely. It is fixable and pretty consistent with results obtained with 7-AAD (although that sample was unfixed). Positive / negative populations looked more clear than with 7-AAD stain.

Experimental Design and Results Summary

Application

Flow Cytometry

Starting Material

Murine bone marrow

Protocol Overview

Incubated 1:100 to 1:1000 (tried many concentrations, ultimately they all worked)

Tips

None

Results Summary

I followed the protocol on the technical data sheet. The first time I did it, it worked OK with 0.5uL dye with surface staining, but the 1:1000 staining in PBS did not (no surface Ab). I tried the protocol a second time with a variety of dye concentrations, ranging from 1:1000 in PBS to 1:100 in PBS (without surface Ab), as well as 0.5uL in PBS with surface Ab. The second time all stains worked, so I'm not quite sure what the problem was the first time. I do think that the 0.5uL staining resulted in bleed over into other channels (I had a problem with some channels, like PacBlue, but no problem with Alexa700). Ultimately, I used the Fixable Viability Dye in PBS, followed by two washes in PBS + FBS, followed by surface staining then fixation in 4% PFA. I thought 1:100 and 1:500 gave the nicest staining (1:100 is the sample shown, but 1:500 looked indistinguishable).

Additional Notes

None

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Summary

The Good

Clear positive and negative populations

The Bad

Requires titration / testing before use.

The Bottom Line

Good option for fixable viability stain. Make sure to test / titrate the dye before using - too little resulted in no staining, too much resulted in bleed over (or some other weird effect on specific channels/stains).

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