Differentiating Viable Vs. Non-Viable Cells In Flow Cytometric Application Using A Fluorochrome Dye.

May 11, 2017

George Washington University, Washington DC
Surgery
Post-doctoral Fellow

Overall

Quality of Results

Ease-of-Optimization

What do these ratings mean?
Write a Review

Company:

Tonbo Biosciences

Product Name:

Ghost Dye™ Violet 510

Catalog Number:

13-0870-T100

Often in multi-color flow cytometric experiments, it becomes imperative that the population of interest is viable which is key to identification of novel differences. In a multi-color panel, using a dye in a channel of Violet laser (405nm) and having emission of 510 (Violet laser with 525/50 filter) makes it easier to identify viable cells without compromising on any standard channels/flurochromes.Thus the viable cells (negative population for Ghost violet 510) can be easily gated out and further multi-color analysis can be done on this viable population which provides more accurate results.

Experimental Design and Results Summary

Application

Flow cytometry

Starting Material

Human PBMC

Protocol Overview

PBMC’s (stimulated vs. non-stimulated) were stained with 1ul (1:100) of Ghost Violet 510 antibody (the same cells were simultaneously stained for a multi-color panel with appropriate antibody dilutions according to the experiment design). Cells were kept for 30 mins. at room temperature. After incubation cells were washed twice with 1XPBS and re-suspended in 500ul of staining buffer. Cells were then analyzed by flow cytometry where mononuclear cells were gated according to FSC and SSC and expression of Ghost violet 510 was gated to be the non-viable fraction. The negative fraction were the viable cells which were gated for further analysis.

Tips

Depending upon the flow machine, filters used, a simultaneous multi-color panel can be used along with Ghost dye 510 for accurate analysis on the viable cell fraction

Results Summary

Basal (non-stimulated) PBMC's showed less er amount of non-viable cells compared to stimulated PBMC's. The viable fraction was gated ( marked as viable cells in the figure) and utilized for further analysis.

DOI or PMID #

N/A

Additional Notes

Choice of flurochrome for other antibodies in the same panel as Ghost dye 510 should be made wisely so that there is no spectral overlap.

Related Categories

Image Gallery

Summary

The Good

A better marker to differentiate between viable and non-viable cells than a surface receptor. Can be used in combination with other flurochrome antibodies for analysis of different markers in a viable cell population.

The Bad

Nothing to mention

The Bottom Line

Differentiating viable and non-viable cells in a multi-color panel using flow cytometry is possible by using ghost violet 510 which gives the option of analysis of expression of different cell markers in a viable population vs non-viable one.

Join the discussion