Identifying Monocytes In Whole Blood By Flow Cytometry Using Anti-HLA-DR-PerCP

February 17, 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:

Anti-human HLA-DR-PerCP antibody

Catalog Number:

347364

We use BD's anti-HLA-DR-PerCP Ab in a panel of antibodies to identify and gate on monocytes in a staining protocol designed for whole blood (human samples). In combination with anti-CD14, BD's anti-human-CD45-APC antibody allows us to identify total monocytes for subsequent breakdown into sub-populations. The antibody provides consistent and easy to interpret staining for identifying our cells of interest..

Experimental Design and Results Summary

Applications

Flow Cytometry

Sample

Human whole blood (100 ul)

Primary Incubation

Incubate 100 ul whole blood with 20 ul of anti-HLA-DR-PerCP 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.

DOI or PMID #

N/A

Additional Notes

N/A

Related Categories

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Summary

The Good

Easy to optimize, reliable and consistent results.

The Bad

Must thoroughly optimize/titrate prior to use.

The Bottom Line

BD's anti-HLA-DR antibody has consistently provided us with clean results using the aforementioned protocol (human whole blood). Once optimized, the protocol in entirety is incredibly simple and time efficient. I would recommend this antibody to anyone staining monocytes using a whole blood approach.

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