Identification of proliferating lymphocytes after PHA-stimulation using an anti-CD25 monoclonal antibody (BioLegend 302617, clone BC96)

July 07, 2016

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

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Quality of Results

Ease-of-Optimization

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Company:

BioLegend

Product Name:

Alexa Fluor® 647 anti-human CD25 Antibody

Catalog Number:

302617

The specific goal of this experiment was to identify proliferating lymphocytes from in vitro phytohemagglutinin stimulation of hum PBMC. This product was chosen because it has been validated by the manufacturer to work for flow cytometry and 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 302617), 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

As expected, a substantial population of PBMC exhibited a high expression of CD25 after 48 hours stimulation with PHA (solid line, lower histogram). Staining was bright, and CD25+ cells were easily distinguishable from negative cells the matched isotype control (broken line, lower histogram). Unstimulated control cells did not show reactivity with anti-CD25, as staining was comparable to the isotype control in these cells (upper histogram, solid line = anti-CD25, broken line = isotype).

Additional Notes

None.

Related Categories

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Summary

The Good

Already optimized for flow cytometry, easy to use, bright staining of stimulated cells.

The Bad

None.

The Bottom Line

Recommended for detection of CD25 expression on stimulated cells using flow cytometry. It is unclear from these results whether this antibody is a good choice for discriminating between CD25-low expressing and -negative events from unstimulated cells.

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