FITC-Conjugated Monoclonal Mouse Anti-Human/Mouse CD3 Antibody (for Flow Cytometry) from BioLegend (clone APA1/1)

May 24, 2016

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

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Company:

BioLegend

Product Name:

FITC anti-human/mouse CD3 epsilon (activation epitope) Antibody

Catalog Number:

362706

This antibody was used to identify CD3+ T cells in human peripheral blood mononuclear cells (PBMC) by intracellular flow cytometry. The epitope recognized by the clone APA1/1 is an activation-dependent intracellular epitope on the CD3 epsilon chain. There was a distinct shift in CD3-expressing lymphocytes, which exhibited bright staining in comparison to CD3-negative cells.

Experimental Design and Results Summary

Applications

Flow Cytometry

Sample

Human PBMC isolated from blood by standard Ficoll density gradient separation

Primary Incubation

FITC anti-human/mouse CD3 antibody (BioLegend 362706), incubated with approximately 1x10^6 cells for 1 hour on ice at 1:20 dilution in a total volume of 100ul PBS+2% FCS, 1mM EDTA, 0.1% sodium azide. Before incubation with antibody, cells were fixed with 4% paraformaldehyde for 20 min on ice, washed, and permeabilized with 0.5% saponin for 30 min on ice.

Blocking Agent

NA

Secondary Incubation

NA

Tertiary Incubation

NA

Detection

FACSCalibur (Becton Dickinson)

Results Summary

This antibody worked well for staining of CD3 on human PBMC by flow cytometry. Staining of CD3+ lymphocytes was bright and they were clearly distinguishable from CD3-negative cells. CD3+CD4+, CD3+CD4-, and CD3-CD4- populations were clearly separated when this antibody was used in combination with an anti-CD4 ab. Some users may wish to titrate this antibody to reduce fluorescence on negative cells so that they resemble unlabelled cells and to extend the number of uses of this antibody. I have used a 1:40 dilution (instead of 1:20) and staining was comparable.

Additional Notes

Cell permeabilization required prior to staining with this antibody. For flow cytometric analysis, gating was performed on the lymphocyte population. Monocytes were negative (not shown).

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Summary

The Good

Directly conjugated with a fluorophore. Minimal optimization. Bright staining of cells. Works with both human and mouse (reported by manufacturer).

The Bad

Fixing and permeabilization of cells required prior to antibody incubation, resulting in a longer cell staining procedure.

The Bottom Line

This antibody performs very well in flow cytometry and is comparable to other anti-CD3 antibodies. If you work with both human and mouse samples, you may want to consider this product over other species specific monoclonals. If fixing and permeabilizing your samples is not an option, you may want to consider a different antibody. Highly recommended!

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