Staining of CD8+ Cells in Human PBMC with Alexa-Fluor 488-Conjugated Rabbit Anti-Human CD8 alpha Monoclonal from Abcam (clone EP1150) for Flow Cytometry

June 02, 2016

Division of BioMedical Sciences, Faculty of Medicine
Memorial University of Newfoundland
Research Assistant

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Company:

abcam

Product Name:

Anti-CD8 alpha antibody [EP1150] (Alexa Fluor 488) (ab203992)

Catalog Number:

ab203992

The primary focus of our research is lymphocyte biology. In order to identify different major and minor lymphocyte subsets in the peripheral blood, we routinely employ flow cytometric staining. The goal of this experiment was to identify CD8+ cells in human PBMC. This product was chosen because it is directly labelled with a fluorophore.

Experimental Design and Results Summary

Applications

Flow Cytometry

Sample

Human peripheral blood mononuclear cells isolated from whole blood by standard Ficoll density gradient separation

Primary Incubation

Cells were fixed in 4% paraformaldehyde for 20 min on ice, washed, and permeabilized with 0.5% saponin for 30 min on ice, and washed again. Rabbit anti-human CD8-alpha antibody (abcam ab203992), was incubated with approximately 1x10^6 cells for 1 hour on ice at 1:400 dilution in a total volume of 100ul PBS+2% FCS, 1mM EDTA, 0.1% sodium azide.

Blocking Agent

N/A

Secondary Incubation

N/A

Tertiary Incubation

N/A

Detection

FACSCalibur (Becton Dickinson)

Results Summary

This antibody worked well in FACS analysis of human PBMC. CD8 expression on gated lymphocytes (side scatter vs forward scatter) is displayed in the histogram. CD8+ cells were sufficiently bright enough to distinguish from CD8-negative events.

Additional Notes

None

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Summary

The Good

Bright staining of cells by flow cytometry. Directly conjugated with a fluorophore, and a couple options are available.

The Bad

Only available in one size. Only verified for use in ICC/IF by the manufacturer.

The Bottom Line

Nice bright staining! Would recommend this to anyone who needs an antibody against human CD8 and for whatever reason has to avoid mouse monoclonals. This directly-labelled rabbit monoclonal performs better than polyclonal abs in my opinion.

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