Anti-CD127 Ab For The Detection & Characterization Of Human T-Cells By Flow Cytometry

February 27, 2017

Clinical Research
Albert Einstein College of Medicine
Pre-doctoral researcher

Overall

Quality of Results

Ease-of-Optimization

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Company:

BD Biosciences

Product Name:

Mouse anti-human CD127-PE

Catalog Number:

557938

Using a whole blood staining protocol, we identify and characterize different T-cell sub-populations by flow cytometry using a panel of antibodies that includes BD's anti-CD127-PE antibody. We stain first for CD3 and CD45 to identify our T-cell populations and subsequently gate on CD4 vs. CD8 to differentiate between the two major populations of T-cells, CD4 and CD8 T-cells. The antibody described here (anti CD127-PE) is one of the stains that we use to identify Regulatory T-cells (Tregs), and once optimized it provides consistent, reliable staining.

Experimental Design and Results Summary

Applications

Flow Cytometry

Sample

Human whole blood (100 ul)

Primary Incubation

Incubate 100 ul whole blood with 5 ul of anti-CD127-PE Ab for 20 minutes on ice.

Blocking Agent

Incubate whole blood with 10 ul of human serum for 10 minutes on ice prior to primary incubation.

Secondary Incubation

Add RBC lysis buffer, vortex, and incubate (covered/no light) at room temperature for 15 minutes.

Tertiary Incubation

Add DAPI at 1:10 and acquire data immediately.

Detection

Positive staining can be detected using a flow cytometer (we use an LSR-II).

Results Summary

Following data acquisition, analyses can be performed using flow cytometry software such as FlowJo. Make sure to run single color and FMO controls with each experiment to ensure the utmost accuracy of results. Here, FMO controls are particularly important because a spectrum of CD127 staining (not just positive vs. negative) is used to differentiate amongs different T-cell sub-populations.

DOI or PMID #

N/A

Additional Notes

N/A

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Summary

The Good

Simple protocol, relatively easy to optimize.

The Bad

Make sure to titrate/optimize antibody with your sample type prior to use on precious samples/materials.

The Bottom Line

I recommend this product for use in flow cytometry assays designed to identify and quantify sub-populations of T-cells from human whole blood samples. When combined, the color combinations (see my other reviews for this protocol) included in the T-cell panel provide clean, consistent results.

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