A Goat Polyclonal Anti-Human CD3-Gamma Antibody (Clone: C-20) That Works Very Well In Flow Cytometry

January 26, 2017

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

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Company:

Santa Cruz Biotechnology

Product Name:

CD3-gamma Antibody (C-20): sc-1125

Catalog Number:

sc-1125

Our lab works with tissues from several species and we are regularly in search of antibodies that work across several species and in different assays in order to reduce laboratory costs. The goal of this experiment was to determine whether this antibody was suitable for use n flow cytometry as FACS is not among the several listed techniques validated by the manufacturer.

Experimental Design and Results Summary

Applications

Flow Cytometry

Sample

Human peripheral blood mononuclear cells

Primary Incubation

Fix cells with 4% formaldehyde for 20 min on ice, wash, and permeabilize with 0.5% saponin (diluted in FACS buffer) for 30 min on ice

Blocking Agent

N/A

Secondary Incubation

Polyclonal goat anti-human/mouse/rat/equine/porcine CD3-gamma antibody (Santa Cruz sc-1125), incubated with approximately 1x10^6 cells for 1 hour on ice at 1:50 (0.4ug) dilution in a total volume of 100ul FACS buffer (PBS+2% FCS, 1mM EDTA, 0.1% sodium azide)

Tertiary Incubation

Alexa fluor 488-conjugated donkey anti-goat IgG (Jackson Laboratories 705-546-147), incubated for 1 hour on ice at 1:400 dilution in a total volume of 100ul permeabilization buffer

Detection

FACSCalibur (Becton Dickinson)

Results Summary

This antibody worked well in flow cytometric analysis of human PBMC. CD3 staining was bright and cells were well separated from negative cells, as shown by the black solid line histogram in the attached figure. Very little background staining compared to isotype control-stained cells (grey-filled histogram) was observed.

DOI or PMID #

N/A

Additional Notes

Gating was performed on the lymphocyte population for analysis.

Related Categories

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Summary

The Good

Bright staining. Little optimization. Antibody works with several species and in several applications other than flow cytometry as confirmed here.

The Bad

Recognizes an intracellular epitope on CD3, thus cell permeabilization is required prior to staining which increases length of assay. Not labelled with a fluorophore, so optimization of secondary antibody may be required.

The Bottom Line

Works very well in flow cytometry when paired with a high quality fluorophore-conjugated secondary antibody. If you are looking for an anti-CD3 antibody that works across several species and in several assays including flow cytometry, then this antibody should be considered.

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