A Good Isotype Antibody for Assessing Non-Specific Antibody Binding on Peripheral CD14+ Monocytes by Flow Cytometry

October 21, 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), PE, eBioscience™

Catalog Number:

12-4714-82

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-CD14 antibody (eBioscience 12-0149-42) to assess the purity of CD14+ monocytes purified from total peripheral blood mononuclear cells.

Experimental Design and Results Summary

Applications

Flow Cytometry

Sample

Purified CD14+ monocytes 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 CD14 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 CD14 to stain affinity purified CD14+ monocytes, no significant non-specific binding of this isotype control antibody was observed (filled histogram). For comparison, CD14 expression analyzed using an anti-CD14 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 non-specific binding. Easy to match with test antibodies because antibody details (isotype, concentration, etc.) are provided by the manufacturer.

The Bad

None

The Bottom Line

Recommended for use as an isotype control in flow cytometry experiments involving PBMC samples or purified CD14+ monocytes when properly matched to a test antibody.

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