A Good Isotype Antibody for Assessing Non-Specific Antibody Binding on Peripheral CD4+ T Cells by Flow Cytometry

October 23, 2019

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

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Company:

eBioscience (Thermo Fisher Scientific - Invitrogen)

Product Name:

Mouse IgG1 kappa Isotype Control (P3.6.2.8.1), PerCP-Cyanine5.5, eBioscience™

Catalog Number:

45-4714-82

Clone Number:

P3.6.2.8.1

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-CD4 antibody (eBioscience 45-0049-42) to assess the purity of CD4+ T cells purified from total peripheral blood mononuclear cells.

Experimental Design and Results Summary

Applications

Flow Cytometry

Sample

Purified CD4+ 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 CD4 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 CD4 to stain affinity purified CD4+ T lymphocytes, very little non-specific binding of this isotype control antibody was observed (filled histogram). For comparison, CD4 expression analyzed using an anti-CD4 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

Very little non-specific binding. Easy to match with your staining antibodies because the manufacturer provides specific details regarding isotype, concentration, etc.

The Bad

None

The Bottom Line

Recommended for use as an isotype control in flow cytometry with PBMC samples or purified CD4+ T lymphocytes when matched to an appropriate test antibody.

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