Satisfactory Option to Stain for PD1 but Not So Bright

May 29, 2020

Charite Strasse 1
Deutsches Rheuma-Forschungszentrum
Research Scientist

Overall

Quality of Results

Ease-of-Optimization

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Company:

BioLegend

Product Name:

APC/Cy7 anti-mouse CD279 (PD-1) Antibody

Catalog Number:

135224

Clone Number:

29F.1A12

In different healthy and pathological contexts CD4 T cells differentiate into a range of specialized interleukin-secreting subsets. In mouse models of aging and autoimmune diseases we study the plasticity and effector functions of CD4 T helper cells. We used anti-PD1 to quantify the number of PD1high CD4 T follicular helper cells in old C57Bl/6J mice and study their cytokine production.

Experimental Design and Results Summary

Applications

Flow Cytometry

Sample

Splenocytes of wild-type 24 months-old C57Bl/6J mouse, incubated in complete culture medium at 37 °C with (Restimulated) or without (unstimulated) PMA/ionomycin for 4h and brefeldin A for the 3 last hours.

Primary Incubation

Surface staining with APC Cy7 anti-PD1 antibody at 1/200, BV650 anti-CXCR5, BV786 anti-CD44 and Pacific Blue anti-CD4 in PBS 0.5% BSA incubated 30min at 4°C. Washed twice in PBS and incubated with L/D fixable aqua dye 20 min at 4°C, then washed twice in PBS.

Blocking Agent

Clone 2.4G2 (Fc block)

Secondary Incubation

Cells are then fixed and permeabilized with commercial kit (30 min at 4°C with fixative, and 15 min at 4°C with permeabilization buffer). Cells are finally incubated with anti-cytokine antibodies in permeabilization buffer for 30 min at 4°C and washed once with permeabilization buffer and once with PBS BSA 0.5%.

Tertiary Incubation

N/A

Detection

Flow cytometry, red laser

Results Summary

I could quantify PD1high CXCR5+ T follicular helper cells amongst CD4+ CD44+ T memory cells of the spleen and measure their cytokine production.

DOI or PMID #

N/A

Additional Notes

N/A

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Summary

The Good

The antibody works fine

The Bad

The staining gives spreading of the positive population into other far red PMT. PD1- and PD1low populations are not well delineated.

The Bottom Line

It is a good option but needs to be combined carefully to avoid too much spreading.

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