CellTrace Violet Cell Proliferation Kit for FACS

June 26, 2017

Columbia University
Microbiology & Immunology
MD/PhD Student

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Company:

Thermo Fisher Scientific

Product Name:

CellTrace™ Violet Cell Proliferation Kit

Catalog Number:

C34571

CD4+ and CD8+ T cell differentiate following activation by TCR stimulation. The differentiation into effector and memory subsets requires cell division, and the acquisition of many phenotypes occurs at certain divisions. We are interested in the process of asymmetric cellular division by T cells and how this can create diversity in cellular progeny. As such our experiments involve tracking the fate of activated T cells and measuring transcription factor expression with respect to cell division. We label naive, unstimulated CD4+ or CD8+ T cells with a cell proliferation dye and subsequently activate in vivo or in vitro with varying stimuli. We use CellTrace Violet (CTV) as an alternative to traditional dyes such CFSE because it has crisper peaks and can be used with GFP-reporter cells.

Experimental Design and Results Summary

Application

Flow cytometry

Starting Material

Mouse CD8+ T cells isolated by magnetic bead enrichment

Protocol Overview

Purified T cells were washed once with dPBS and resuspended in dPBS containing 2.5 micromolar CellTrace violet (1:2000 dilution from a stock solution of 5mM prepared in DMSO according to the manufacturer's instructions). Cells were then incubated at 37C for 20 minutes followed by 2 washes with excess complete media containing serum to quench the labeling reaction, e.g. 10% FBS in RPMI. After labeling, T cells were used for in vitro or in vivo activation as usual.

Tips

CellTrace Violet can be freeze/thawed without a decrease in labeling efficiency/intensity

Results Summary

In this experiment, CD8+ T cells were labeled with CellTrace Violet. Roughly 20% of the cells died during the labeling process, in line with, or better than, the average cell loss that we see from other proliferation dyes. Cells were then activated with anti-CD3/CD28 stimulation in vitro and harvested at 60 hours post-stimulation. Samples were analyzed by multi-color flow cytometry on a BD Fortessa. Crisp division peaks were observed, with the cells undergoing anywhere from 1-4 divisions. Gates around individual divisions could be drawn readily. Fluoresence of the division dye was preserved following fixation/permeablization with the eBioscience Foxp3 transcription factor staining kit.

DOI or PMID #

N/A

Additional Notes

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Image Gallery

Summary

The Good

Bright, crisp division peaks for at least 7 generations of cell division

The Bad

Loss of some cells (20% death) with every labeling reaction.

The Bottom Line

CellTrace Violet is a very bright proliferation dye that allows for easy, clear gating of individual cell divisions across many generations of proliferation.

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