Good Staining in Flow Cytometry with Mouse Monoclonal (C8/468 + C8/144B) to Human CD8 alpha

May 20, 2016

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

Overall

Quality of Results

Ease-of-Optimization

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Company:

Abcam

Product Name:

Anti-CD8 alpha antibody [C8/468 + C8/144B] (ab199016)

Catalog Number:

ab199016

The main area of focus of our lab is lymphocyte biology, with a focus on T cell biology. Our goal in using this product was to identify CD8+ cells in human peripheral blood mononuclear cells. This particular product was chosen because it contains two different monoclonal antibodies recognizing two different epitopes instead of only one as is the case for the majority of other monoclonal abs. We also selected this antibody because of the wide variety of applications that it is reported to work in by the manufacturer. Even though we were initially using this ab in flow cytometry, we wanted the flexibility to move to another application if necessary.

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. Mouse anti-human CD8-alpha antibody (abcam ab199016), 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

Alexa Fluor 647-conjugated donkey anti-mouse IgG (H+L) (Jackson 715 605 151), 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

CD8-positive cells were stained very bright and could easily be distinguished from negative cells. Background staining was minimal and comparable to the isotype control.

Additional Notes

For flow cytometric analysis, gating was performed on the lymphocyte population. Monocytes were negative (not shown).

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Summary

The Good

Bright staining of CD8+ cells. Does not need much optimization. Suitable for use in a wide variety of applications in addition to flow cytometry.

The Bad

Intracellular epitope so fixing and permeabilization is required which require additional optimization and lengthen staining time. No fluorphore-conjugated version of this antibody available for purchase. Only available in one test size.

The Bottom Line

This is a very good CD8 antibody for flow cytometry with the added bonus of a manufacturer guarantee to work with additional applications varying from western blot to IHC. If you are looking for a CD8 antibody that performs well in FACS with the future possibility of moving to different applications, this should be considered. However, the addition of fixation, permeabilization, and secondary ab incubation significantly increases staining time in comparison to a fluorophore-conjugated ab that recognizes an extracellular epitope.

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