Very Bright Staining

March 16, 2018

Immunology
Columbia University
Graduate Student

Overall

Quality of Results

Ease-of-Optimization

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Company:

Biolegend

Product Name:

PE anti-human HLA-A,B,C Antibody

Catalog Number:

311405

Class I antigen presentation is utilized by virtually every cell in the body not only to present foreign antigens to elicit immune responses, but also is used to present self-peptides to prevent destruction by NK cells. Many cancers have evolved to downregulate class I presentation will upregulating "decoys" to prevent NK mediated destruction. As part of our screen for potent immunomodulatory agents with therapeutic application in cancer, we look for compounds that can increase Class I presentation in human cancer lines, in the hopes that this allows for better recognition and destruction by cytotoxic CD8s.

Experimental Design and Results Summary

Applications

Flow Cytometry

Sample

PC3 Human Prostate Adenocarcinoma Lines

Primary Incubation

1:100 for 20 mins at RT

Blocking Agent

N/A

Secondary Incubation

N/A

Tertiary Incubation

N/A

Detection

Flow Cytometry

Results Summary

PC3 cells robustly express Class I on their surface at baseline, so it was important to set the voltage after running these samples in order to ensure the voltage was low enough that they would not be off-scale. On the BD Celesta, this meant using a voltage that shifted the median flourescence of the unstained sample to about 10^2. The staining was bright, and compatible with BV421 as well as APC/Cy7. Analysis was done based off live cells as assayed by using a viability die. IFN-y is a known upregulator of class I, and was used as a positive control. In the PC3 cell line, HLA-ABC was upregulated after stimulation with IFN-y.

DOI or PMID #

N/A

Additional Notes

Higher baseline levels of HLA-ABC may require even more diluted amounts

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Summary

The Good

Very bright, can be diluted further to save antibody

The Bad

Takes some optimization

The Bottom Line

A little goes a long way with this antibody. An understanding of Class I baseline expression will really help in optimizing this antibody - if a line expresses a lot, then the antibody should be diluted much more. Additionally, as a positive control, if lines are very sensitive to IFN-y (the prostate lines I have worked with are not), further optimization will be needed to make sure it stays on scale. I recommend running the unstained (negative control), sample, and positive control first to set the voltage before starting to record data.

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