CD3 Staining Of Human PBMC For Flow Cytometry Using A Rat Anti-Human CD3:FITC Antibody (Clone CD3-12)

April 04, 2017

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

Overall

Quality of Results

Ease-of-Optimization

What do these ratings mean?
Write a Review

Company:

Bio-Rad (Formerly AbD Serotec)

Product Name:

CD3 Antibody CD3-12 : Rat anti Human CD3:FITC

Catalog Number:

MCA1477FT

One of the main research areas of our lab concerning lymphocyte biology with a particular focus on the function of T cell subsets in diseased individuals. In order to successfully study T cell subsets from patient samples using flow cytometry, quality antibodies are a necessity. Our goal in using this product was to identify a quality anti-CD3 antibody for flow cytometry. This particular antibody was chosen because (a) it is directly labelled with a fluorophore thus eliminating the use of a secondary-fluorophore conjugated antibody and (b) it is reported by the manufacturer to be cross-reactive with several other species, thereby allowing use of the same reagents for different biological samples if required in the future.

Experimental Design and Results Summary

Applications

Flow Cytometry

Sample

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

Primary Incubation

Cells were fixed in 4% paraformaldehyde for 20 min on ice, washed, and permeabilized with 0.5% saponin for 30 min on ice, and washed again.

Blocking Agent

N/A

Secondary Incubation

Approximately 1x10^6 cells were incubated on ice for 1 hour in the dark with Rat anti Human CD3:FITC (Bio-Rad MCA1477FT; clone CD3-12) at a 1:10 dilution in a total volume of 100ul PBS+2% FCS, 1mM EDTA, 0.1% sodium azide.

Tertiary Incubation

N/A

Detection

FACSCalibur (Becton Dickinson)

Results Summary

This antibody worked well in flow cytometric analysis of human PBMC. CD3+ cells were fairly well separated from CD3- cells based on fluorescence intensity, making both populations easily distinguishable from each other (open histogram). CD3- cells resembled cells stained with a matched isotype control (grey filled histogram).

DOI or PMID #

N/A

Additional Notes

For flow cytometric analysis, gating was performed on the lymphocyte population identified by forward vs side scatter.

Related Categories

Image Gallery

Summary

The Good

Directly labelled with a fluorophore. Manufacturer provides recommended dilutions for testing, thus minimizing optimization time. Works with several species, thus allowing the same reagents to be used if you are performing both animal and human studies/experiments simultaneously.

The Bad

This antibody recognizes an Intracellular epitope, thus cell fixation and permeabilization are required which results in longer assay times and may not be compatible in multi-staining experiments involving several antibodies.

The Bottom Line

This antibody stained CD3 fairly well on human PBMC for flow cytometry. Separation between CD3- and CD3+ cells was just OK. There are other products available that result in better separation between the two populations. The benefit of this product is that it is reported by the manufacturer to work with several species. Therefore, if your lab simultaneously utilizes several species for your studies, this antibody might be appealing.

Share your experience with other scientists. Write a Review! »

Join the discussion