Great Cellular Dye to Monitor Proliferation

January 10, 2019

Johns Hopkins School of Medicine
Oncology
Postdoctoral Fellow

Overall

Quality of Results

Ease-of-Optimization

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Company:

Thermo Fisher Scientific

Product Name:

Cell Proliferation Dye eFluor450 (ef450)

Catalog Number:

65-0842-85

As an immunologist, we routinely assess cell proliferation via flow cytometry. CFSE was the original dye used to perform this task but new dyes in different channels exist such as ef450 for multi-color panels. This dye binds to protein in naive T cells and dilutes itself as cells divide evenly for a maximum of 6–7 divisions before it's no longer visible. The dye is very consistent.

Experimental Design and Results Summary

Application

Cell proliferation by flow cytometry

Starting Material

Murine T cells

Protocol Overview

We reconstituted the dye as mentioned (165 µL DMSO/vial). Aliquot the dye and re-freeze for future uses. Obtain single-cell suspension of T cells in warm 37°C PBS. The cells are adjusted to 10e6/mL in the PBS then stained with 1:1000 of the ef450 dye for 10 minutes at 37°C. The reaction is immediately quenched with cold media containing 10% serum on ice for 5 minutes then washed once before proceeding with experiment.

Tips

Make sure counts are close to 10e6/ml for consistent results along with using warm PBS.

Results Summary

We are able to visualize cell division upon stimulation of CD8 T cells within 72 hrs.

DOI or PMID #

N/A

Additional Notes

N/A

Image Gallery

Summary

The Good

Very consistent and reliable staining ensures uniform peaks. Viability after staining is very good also unlike CFSE.

The Bad

None whatsoever

The Bottom Line

Great dye to monitor cell proliferation using flow cytometry. Way better than CFSE since peaks are always uniform and consistent.

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