Analysis of T-cell Central Memory Population

June 19, 2015

George Washington University, Washington DC
Surgery
Post-doctoral Fellow

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Company:

BioLegend

Product Name:

CD44 PECy7 (anti-mouse human antibody) and CD62L APC

Catalog Number:

103030 (CD44 PECy7) and 104412 (CD62L)

A small fraction of Naive T cells upon primary exposure to foreign or self antigens differentiate to form a population called T central memory cells (TCM). Upon re-exposure to the same antigen the TCM undergoes differentiation and divides rapidly to form effector memory cells (TEM) T cells with a phenotype of CD62L positive and CD44high has been well referred as this fraction of central memory cells (Klebanoff CA, Gattinoni L, Restifo NP. CD8+ T-cell memory in tumor immunology and immunotherapy. Immunol Rev. 2006;211:214–224). Analysis of TCM is relatively simple by utilizing detection of specific surface receptor molecules that it possess while it is TCM (CD44high/CD62L+) and changes phenotype rapidly (loses CD62L) upon activation/stimulation. Using flow cytometry, the fraction of TCM at the basal level and after stimulation/ activation can be easily analyzed.

Experimental Design and Results Summary

Application

Flow Cytometry

Starting Material

T cells; splenocytes

Protocol Overview

CD8+T cells were isolated from mice spleens expressing a specific Human TCR (transgenic mice). 20,000 cells were incubated in each well of a 96 well round bottom plate with staining buffer containing an antibody cocktail containing CD44PECy7 (1:100 dilution) CD62L APC (1:50 dilution) and CD8 FITC (1:100 dilution). Cells were incubated for 30 minute preferably in the dark and on ice. After completion of the incubation period, cells were washed twice with PBS and resuspended in 200-300ul of staining buffer (containing 2% FBS and 0.05%sodium azide) and flow cytometry was performed. Cells were initially gated for CD8 positive cells and then fraction of CD44 high/ CD62L+ cells was estimated.

Tips

CD44 PECy7 has a very strong fluorescence and thus standardization of its dilution is important.

Results Summary

Distinct populations of CD44high/CD62L+ is found at the basal level (TCM population) which decreases after stimulation/ activation.

Additional Notes

Analysis can be modified according the need to identify TCM, TEM or just effector population.

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Summary

The Good

Quick, easy and reliable methodology. Results are repeatable.

The Bad

Absolutely nothing if stringent protocol is maintained.

The Bottom Line

Good, reliable and relatively inexpensive method using flow cytometry to analyze TCM/TEM population.

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