Determining CD25 expression on PHA-stimulated lymphocytes by flow cytometry using an anti-CD25 monoclonal antibody (BioLegend 356127, clone M-A251)

July 15, 2016

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

Overall

Quality of Results

Ease-of-Optimization

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Company:

BioLegend

Product Name:

Alexa Fluor® 647 anti-human CD25 Antibody

Catalog Number:

356127

The primary goal of this experiment was to identify proliferating lymphocytes based on CD25 expression following in vitro stimulation with phytohemagglutinin. This antibody was chosen because (a) it is validated for use in flow cytometry by the manufacturer and (b) is directly-labelled with a fluorophore.

Experimental Design and Results Summary

Applications

Flow Cytometry

Sample

Human PBMC isolated by Ficoll and stimulated with 5ug/ml phytohemagglutinin (PHA) for 48 hrs in cell culture

Primary Incubation

Alexa Fluor 647 anti-human CD25 antibody (BioLegend 356127), 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

N/A

Secondary Incubation

N/A

Tertiary Incubation

N/A

Detection

FACSCalibur (Becton Dickinson)

Results Summary

CD25 expression was upregulated on a significant proportion of PHA-stimulated human lymphocytes (red, lower dot plot). Staining was bright and CD25-expressing cells were easily distinguished from negative cells and cells labelled with a matched isotype control (black, lower dot plot). Furthermore, cells which did not express CD25 resembled the isotype control.As expected, CD25 expression was minimal on freshly isolated unstimulated control PBMC. Only a small percentage of cells expressing low amounts of CD25 were detected on unstimulated PBMC (red, upper dot plot) compared to the isotype control (black, upper dot plot).

Additional Notes

None

Related Categories

Image Gallery

Summary

The Good

Bright staining by flow cytometry, directly-labelled with a fluorophore, little to no optimization required.

The Bad

None

The Bottom Line

This antibody is highly recommended for measuring CD25 expression on human lymphocytes. Staining is bright, allowing identification of both CD25low and CD25high expressing cells. In addition, it comes ready to use with little optimization required.

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