Strong Staining, Low Background

March 14, 2018

Immunology
Columbia University
Graduate Student

Overall

Quality of Results

Ease-of-Optimization

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Company:

Biolegend

Product Name:

Brilliant Violet 421™ anti-human CD274 (B7-H1, PD-L1) [29E.2A3] 25 tests

Catalog Number:

329713

The goal of this experiment was to look at how various pharmacological agents could affect the expression of CD274/PD-L1 on the surface of human cancer cell lines. This is done in order to better understand both how surface expression is regulated as well as how different classes of compounds could affect this regulation at transcription, translation, or through post-translational modifications. This antibody was used to assess levels of PD-L1 on several cell lines using flow cytometry on a BD Celesta instrument.

Experimental Design and Results Summary

Applications

Flow Cytometry

Sample

PC3 Human Prostate Adenocarcinoma Cell Line

Primary Incubation

1:50, 20 minutes at RT in dark

Blocking Agent

N/A

Secondary Incubation

N/A

Tertiary Incubation

N/A

Detection

Flow Cytometry

Results Summary

Approximately 2x10^5 cells were stained in a 96 well plate with 50 uL of antibody mix (1:50 in PBS). Analysis was based on live cells only (gated based on viability dye). In this cell line, DMSO treatment at 1:10k showed similar expression levels of PD-L1 as nontreated cells. IFN-y, a known upregulator of PD-L1, increased the expression of PD-L1 almost fivefold. We have repeated this assay with the DU145 line and found this trend to hold true as well.Cells were not fixed prior to assay.

DOI or PMID #

N/A

Additional Notes

N/A

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Summary

The Good

Easy to use, consistent results across cell lines

The Bad

Cost, since each test requires a large amount of antibody

The Bottom Line

This is a great antibody for detecting PD-L1 on human cells. The fluorescence is bright and easy to compensate. Dilution at 1:50 yields good results for most cell lines I have tried. I have also tried 1:100, but the separation of peaks (IFN-y induced vs no tx) is poor for cell lines that express low levels of PD-L1 at baseline.

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