Okay But Not Great Staining of CD8+ T Cells in Human PBMC by Flow Cytometry Using a Polyclonal Rabbit Anti-Human CD8 alpha Antibody

May 31, 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 (ab187366)

Catalog Number:

ab187366

The general aim of our research is the study of lymphocyte biology, with a particular focus on T cells. In order to study the expression of various molecules on cytotoxic T cells from peripheral blood and in tissues, we first require the identification of CD8+ T cells. This polyclonal anti-CD8a antibody was initially chosen because it is reported by the manufacturer to work in several different applications including IHC, ICC/IF, flow cytometry, western blot, and immunoprecipitation.

Experimental Design and Results Summary

Applications

Flow Cytometry

Sample

Human PBMC isolated from 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 polyclonal antibody (abcam ab187366), was incubated with approximately 1x10^6 cells for 1 hour on ice at 1:20 dilution (1.0ug antibody) in a total volume of 100ul PBS+2% FCS, 1mM EDTA, 0.1% sodium azide.

Blocking Agent

NA

Secondary Incubation

Dylight 649-conjugated goat anti-rabbit IgG (H+L) (Jackson 111-495-003), incubated for 1 hour on ice in the dark at 1:800 dilution in a total volume of 100ul PBS+2% FCS, 1mM EDTA, 0.1% sodium azide.

Tertiary Incubation

NA

Detection

FACSCalibur (Becton Dickinson)

Results Summary

Although two populations were observed in both the flow cytometry density plot and histogram, presumably corresponding to CD8+ and CD8- T cells, there was little separation between the two in terms of fluorescence, thus making it difficult to fully distinguish the separate populations. Cells incubated with secondary antibody alone (shaded histogram) had considerably less background compared to the supposed CD8- population. Further optimization may be required to improve results (i.e. primary and secondary antibody concentrations).

Additional Notes

For flow cytometric analysis, gating was performed on the lymphocyte population.

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Summary

The Good

Guaranteed by the manufacturer to work in several applications.

The Bad

Not great staining of CD8+ cells making it difficult to distinguish different populations. Requires optimization. Not directly labelled. Intracellular epitope which increases the length of staining for flow cytometry.

The Bottom Line

In our experience, this antibody performs poorly in flow cytometry compared to other anti-human CD8a antibodies. Look elsewhere if you need to measure CD8 expression or distinguish CD8+ T cells from other lymphocyte populations by flow cytometry.

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