Detection of CD86 via FITC-labeled antibody in mice peritoneal macrophages

May 22, 2015

Pharmaceutical Sciences
University of Michigan
Research Fellow

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Company:

BD Biosciences

Product Name:

FITC Rat Anti-Mouse CD86 Clone GL1 (RUO)

Catalog Number:

553691

Browse Similar Products: CD86 Antibodies

This antibody was used (amongst others) for the detection of CD86 expression in peritoneal macrophages to detect classical or alternative activation of macrophages upon addition of crystallized drug deposits of clofazimine. In order to confirm that only peritoneal macrophages were analyzed for the detection of CD86 expression, dual-labeled cells were analyzed via using F4/80 antibody on the same cell population. See review of this antibody here - https://www.biocompare.com/Product-Reviews/171258-Fantastic-Product/?arev=true

Experimental Design and Results Summary

Applications

Flow Cytometry

Sample

Peritoneal exudate obtained from mice (C57Bl6 strain). See protocol for obtaining peritoneal exudate here - http://www.jove.com/video/1488/isolation-of-mouse-peritoneal-cavity-cells

Primary Incubation

Since the experiment involved dual-labeling both F4/80 and CD86, antibodies were added sequentially. F4/80 details (1 ug/100,000 cells; 1:10 volumetric ratio of antibody to cells; 30 minutes; 4 deg C in dark)

Blocking Agent

Standard Fc block solution - (Affymetrix ebioscience - Cat No. - 16-0161-86). 1 ug per 300,000 cells

Secondary Incubation

CD86 incubation details - (Same as F4/80 as above)

Tertiary Incubation

NA

Detection

MoFlo Astrios (BD), FITC fluorescence

Results Summary

The antibody worked pretty well and CD86+ macrophages (post F4/80 gating) were easily detected relative to isotype (See image below)

Additional Notes

Only Flow cytometry application is reviewed. Other applications were not tested.

Related Categories

Image Gallery

Summary

The Good

Easy use, economical for this application. No cross-over with the other fluorescence channel.

The Bad

FITC signal may be weak, hence fluorochromes with higher intensity and less photobleaching, if available, may be used.

The Bottom Line

This antibody can be used to detect classically activated macrophages. In applications where mixed cell populations are the norm, such as in the peritoneal exudate, use of other macrophage-specific markers (such as F4/80) should be combined with this antibody.

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