Decently Bright, Difficult to Compensate

February 18, 2019

Immunology
Columbia University
Graduate Student

Overall

Quality of Results

Ease-of-Optimization

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Company:

BioLegend

Product Name:

Brilliant Violet 510™ anti-mouse CD8a

Catalog Number:

100752

This antibody was used for delineating CD8+ cells from murine tumors for further downstream analysis (activation, proliferation, naive vs. memory, etc). As controls, spleens and lymph nodes were harvested and stained in order to set gates. When looking at CD4 vs CD8, there was a clear CD8+ population using this antibody. The population was bright at 1:500 and was stable when fixed, even after 6 days. When using this as a single-stain control, there was considerable bleed into BV605 and BV650 which made compensation difficult.

Experimental Design and Results Summary

Applications

Flow Cytometry

Sample

Murine Spleen (Control)

Primary Incubation

20 minutes (Surface Stain), RT

Blocking Agent

N/A

Secondary Incubation

N/A

Tertiary Incubation

N/A

Detection

BV510 (525/40-nm)

Results Summary

Spleen was isolated from tumor bearing mice as controls for gating. Following mechanical dissociation and red blood cell lysis, approximately 5x10^5 million cells were plated and stained with viability dye, CD45, TCRb, CD4, and CD8 (1:500) in staining buffer. The primary incubation was for 20 minutes at room temperature. After a PBS wash, the cells were then fixed and permeabilized for 45 minutes at room temperature. Following intracellular staining for cytokines, cells were then washed in perm buffer and then resuspended in PBS and stored for 6 days at 4°C prior to being run on a BD FacsCelesta flow cytometer. Gating was determined based on lymphocyte size gate on live cells that were CD45+TCRb+. All analyses were performed using FlowJo software after data collection.

DOI or PMID #

N/A

Additional Notes

For a cleaner population, gate on CD3/CD45/TCRb first

Related Categories

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Summary

The Good

Pretty bright, clear CD8/CD4 populations

The Bad

Difficult to compensate

The Bottom Line

When running panels of 12+ colors on mouse lymphocytes, this is a very good antibody for marking CD8+ cells. It is fairly bright when used at 1:500 and remains bright for at least 6 days after staining when fixed. It bleeds into BV605 and BV650 a little heavily so good compensation requires bright and good quality single stains in all three channels. Easy to use and doesn't really require much titration to use. At 1:500, it's also very economical.

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