Labeling Human Primary PBMCs for CCR7

July 01, 2016

Anaesthesiology
LMU Munich
Researcher

Overall

Quality of Results

Ease-of-Optimization

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Company:

Biolegend

Product Name:

Brilliant Violet 421™ anti-human CD197 (CCR7) Antibody

Catalog Number:

353207

In order to characterize human Memory T Cells, we set out to label human PBMCs with CCR7 and other memory and T cell markers such as CD62L, CD3, CD4, CD8, CD45RO and CD45RA.

Experimental Design and Results Summary

Applications

Flow Cytometry

Sample

Primary Human PBMC

Primary Incubation

5µl per 1 million cells, incubation 20 Mins on ice in the dark, wash 3 times with FACS buffer (PBS, 1% BSA, 0,001% Na Azide)

Blocking Agent

Tru Stain FcX Receptor blocking solution (Biolegend)

Secondary Incubation

None

Tertiary Incubation

None

Detection

Attune Acoustic Focusing Cytometer

Results Summary

This antibody allows clear identification of CCR7-positive cells. Together with other memory cell markers, this antibody allows a robust, reproducible and reliable quantification of memory cells. Cryopreservation may significantly affect CCR7-staining, better use fresh cells.

Additional Notes

Be sure to Fc-Block and also use fresh cells, not cryopreserved ones.

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Summary

The Good

Quick protocol, suggested antibody concentration works well with PBMCs, rather small emission spectrum

The Bad

Antibody conjugate is pretty expensive, extensive washing may be needed, cryopreservation may affect staining.

The Bottom Line

Easy to use, reproducible, robust results, expensive, but recommendable Be careful with cryopreservation.

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