A Very Good Antibody To Measure TIM-3 Expression On T Cells From Peripheral Blood By Flow Cytometry

March 24, 2017

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:

BD Biosciences

Product Name:

PE Mouse Anti-Human TIM-3 (CD366) Clone 7D3

Catalog Number:

563422

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 T cell exhaustion markers. This particular antibody was chosen since it is (a) directly labelled with a fluorophore, and (b) comes pre-diluted reeady to use for flow cytometry.

Experimental Design and Results Summary

Applications

Flow Cytometry

Sample

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 T cell exhaustion markers. This particular antibody was chosen since it is (a) directly labelled with a fluorophore, (b) already titrated for use in flow cytometry, and (c) given BioLegend's exceptional reputation.

Primary Incubation

Approximately 1x10^6 cells were incubated on ice for 1 hour in the dark with PE Mouse Anti-Human TIM-3 (CD366) antibody (563422; clone 7D3) 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 expressed primarily on CD3+ T cells after T-cell stimulation with PMA/Ionomcyin. ~21% of CD3+ cells expressed TIM-3 after overnight stimulation with PMA/Ionomycin compared to <1% in unstimulated control cells. Compared to a matched isotype control, TIM-3+ cells were easily identifiable.

DOI or PMID #

N/A

Additional Notes

N/A

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Summary

The Good

Directly labelled with a fluorophore. Already titrated by the manufacturer before shipping so very little optimization is required.

The Bad

None

The Bottom Line

I highly recommend this antibody for use in flow cytometry to measure TIM-3 expression on human blood cells. Given the relatively low, or lack of expression on resting/non-stimulated T-cells as observed here, you may want to have a positive staining control when measuring TIM-3 expression on biological samples to ensure that you are getting adequate staining as observed with our PMA/Ionomycin-stimulated PBMC.

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