A Good Isotype Antibody for Assessing Non-Specific Antibody Binding on Peripheral CD8+ T Cells by Flow Cytometry

October 17, 2019

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

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Quality of Results

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Company:

eBioscience (Thermo Fisher Scientific - Invitrogen)

Product Name:

Mouse IgG2a kappa Isotype Control (eBM2a), Alexa Fluor 488, eBioscience™

Catalog Number:

53-4724-80

Clone Number:

eBM2a

Our research involves the study of immune cell phenotypes in different disease states with a focus on viral infection. This antibody was used to assess non-specific antibody binding and used in combination with the anti-CD8 antibody (eBioscience 53-0086-42) to assess the purity of CD8+ T cells purified from total peripheral blood mononuclear cells.

Experimental Design and Results Summary

Applications

Flow Cytometry

Sample

Purified CD8+ T cells from human PBMC by magnetic bead-based positive selection

Primary Incubation

2.0x10^5 cells were stained with the equivalent amount of antibody used to stain for CD8 in a total staining volume of 100ul autoMACS running buffer (Miltenyi Biotec) for 30 minutes on ice in the dark.

Blocking Agent

10 min on ice with human Fc receptor blocking reagent (Miltenyi Biotec) prior to primary incubation with fluorochrome-labelled antibody

Secondary Incubation

N/A

Tertiary Incubation

N/A

Detection

Becton Dickinson FACSCalibur flow cytometer

Results Summary

When used at an equivalent concentration as a test antibody against CD8 to stain affinity purified CD8+ T cells, no significant non-specific binding of this isotype control antibody was observed (filled histogram). For comparison, CD8 expression analyzed using an anti-CD8 antibody of the same isotype and labelled fluorophore is shown (open histogram).

DOI or PMID #

10.1186/s12865-015-0113-0

Additional Notes

N/A

Image Gallery

Summary

The Good

No evidence of significant non-specific binding. The details of the antibody (isotype, concentration, etc) are clearly stated by the manufacturer, making it easy to match this isotype control to your test antibody staining.

The Bad

None

The Bottom Line

Highly recommended for use as an isotype control in flow cytometry experiments involving PBMC samples or purified CD8+ T cells when properly matched to a test antibody.

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