Anti-CD3-FITC Ab for the detection of human T-cells by flow cytometry

February 22, 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 CD3-FITC

Catalog Number:

555332

We use the anti-CD3-FITC Ab in a whole blood staining protocol designed to identify, characterize, and quantify T cells by flow cytometry. When plotted against CD45 expression, BD's anti-human-CD3-FITC antibody provides perfect separation of T cells from other leukocytes present in our samples. For all of our studies to date, the staining has been consistent and reliable.

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-CD3-FITC 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. Once leukocytes have been identified, T-cells can be gated on based on CD3+CD45+ staining.

DOI or PMID #

N/A

Additional Notes

N/A

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Summary

The Good

Relatively inexpensive, easy to optimize, reliable and consistent results.

The Bad

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

The Bottom Line

I highly recommend this antibody for T-cell staining protocols using whole blood for flow cytometry applications. The antibody was incredibly easy to titrate/optimize and has continually provided us with clean, consistent results.

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