Highly Recommended Anti-TIM-3 Antibody For Staining Of Human Peripheral Blood Mononuclear Cells By Flow Cytometry

March 28, 2017

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:

PE anti-human CD366 (Tim-3) Antibody

Catalog Number:

345006

One of the interests of our lab is T-cell exhaustion in the context of viral infection and cancer. Quality reagents are required to study T-cell exhaustion cell markers by flow cytometry given their relatively low baseline expression under normal conditions as well as the relatively low frequency of expressing cells under physiological conditions. The purpose of this experiment was to establish an in vitro assay to measure the expression of TIM-3 on peripheral blood cells. This particular antibody was chosen since it is (a) directly labelled with a fluorophore, (b) already titrated for use in flow cytometry, and (c) our previous good experience with BioLegend antibodies.

Experimental Design and Results Summary

Applications

Flow Cytometry

Sample

Human peripheral blood mononuclear cells stimulated overnight with 25ng/ml phorbol 12-myristate 13-acetate and 100ng/ml ionomycin

Primary Incubation

Approximately 1x10^6 cells were incubated on ice for 1 hour in the dark with PE anti-human CD366 (Tim-3) Antibody (345006; clone F38-2E2) at a 1:20 dilution in a total volume of 100ul PBS+2% FCS, 1mM EDTA. At the same time, the cells were stained with a mouse-anti-human CD3 antibody.

Blocking Agent

N/A

Secondary Incubation

N/A

Tertiary Incubation

N/A

Detection

BD FACSCalibur

Results Summary

TIM-3 expression was upregulated on both CD3+ and CD3- cells after stimulation with PMA/Ionomcyin. Approximately 15% of CD3+ cells became TIM-3+ after overnight stimulation with PMA/Ionomycin compared to <1% in unstimulated control cells. In addition, the number of CD3- TIM-3+ cells doubled after stimulated compared to control cells. Compared to a matched isotype control, TIM-3 staining was relatively bright and obvious.

DOI or PMID #

N/A

Additional Notes

N/A

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Summary

The Good

Directly labelled with a fluorophore. Already diluted by the manufacturer prior to shipping, thus very little optimization required.

The Bad

None

The Bottom Line

This antibody was excellent in staining TIM-3+ cells after T-cell stimulation of human peripheral blood cells for flow cytometry. Users may want to include a positive staining control (i.e. TIM-3 expressing cell line, stimulated/activated cells) in their experiments to compliment their biological samples, as TIM-3 expression can be low, or non-existent as observed here, on healthy unstimulated/resting lymphocytes.

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