FITC-Conjugated Monoclonal Mouse Anti-Human CD3 Antibody (for Flow Cytometry) from BioLegend (Clone OKT3)

May 18, 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 CD3 Antibody

Catalog Number:

317306

This antibody was used to identify CD3+ T cells in human peripheral blood mononuclear cells (PBMC) by flow cytometry. Bright staining was observed by FACS analysis, which allowed clear identification of CD3-positive and -negative cells.

Experimental Design and Results Summary

Applications

Flow Cytometry

Sample

Human peripheral blood mononuclear cells isolated from whole blood by standard Ficoll density gradient separation.

Primary Incubation

FITC anti-human CD3 antibody (BioLegend 317306), 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.

Blocking Agent

NA

Secondary Incubation

NA

Tertiary Incubation

NA

Detection

FACSCalibur (Becton Dickinson)

Results Summary

This antibody worked excellent in FACS analysis of CD3 surface expression on human PBMC. CD3 staining was bright and positive cells were well separated from negative cells. Minimal background staining was observed compared to the unlabelled control. Some users may want titrate this antibody beyond 1:20 so that negative cells resemble unlabelled cells.

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. Minimal background. Directly-conjugated with FITC, thus easy and quick staining protocol. Surface epitope, so no fixation/permeabilization required. Very affordable!

The Bad

Nothing that I can think of.

The Bottom Line

This is an excellent antibody for surface staining of CD3 on human PBMC. CD3-expressing cells are easily differentiated from negative cells.

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