Stains Ebi3 Subunit In B Cells

December 19, 2016

BlueskyReddit
Immunology
Duke University
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Company:

R&D Systems

Product Name:

Mouse IL-27/IL-35 EBI3 Subunit PerCP-conjugated Antibody

Catalog Number:

IC18341C

The goal of this research study was to evaluate mouse splenic B cell activation molecule expression after short-term in vitro stimulation with different mitogenic stimuli. This product was selected because it was the only available mAb that was specific for the Ebi3 subunit, as opposed to reacting with it in heterodimeric form, and because it is conjugated to a fluorophore usable by the available flow cytometer.

Experimental Design and Results Summary

Applications

Flow Cytometry

Sample

Mouse Splenocytes

Primary Incubation

Isolate splenocytes. Wash cells once in FACS buffer (PBS plus 1% BSA or serum). Block Fc receptors and stain for viability in PBS for 20 minutes on ice.

Blocking Agent

Fc block

Secondary Incubation

Wash cells once in FACS buffer. Stain with isotype control or CD19 antibody (1:500 dilution, 100 uL per million cells) for 20 minutes on ice.

Tertiary Incubation

Wash cells once in FACS buffer. Permeabilize cells at room temperature for 1 hour or overnight. Wash cells in permeabilization buffer Stain with isotype control or mouse Ebi3 antibody (1:100 dilution in permeabilization buffer, 100 uL per million cells) for 20 minutes on ice. Wash cells once in permeabilization buffer. Resuspend cells in 100-250 uL PBS containing 1.5% paraformaldehyde.

Detection

Flow Cytometer

Results Summary

Following stimulation with LPS for 24 hours and then in the last 5 hours either monensin alone (panels second from the left) or PMA, ionomycin, and monensin (first and third panels from the left), Ebi3 expression was clearly upregulated with activation (middle panel, shaded histogram in far right panel) relative to isotype control (first panel from the left, empty histogram in far right panel).

DOI or PMID #

N/A

Additional Notes

N/A

Related Categories

Image Gallery

Summary

The Good

Shows shift in Ebi3 relative to isotype control.

The Bad

Because it is a PerCP antibody, all work has to be done in the dark, including running the cells on the cytometer, because PerCP is very unstable.

The Bottom Line

Useful for showing shifts in Ebi3 expression, but is more difficult than other antibodies to work with due to the PerCP conjugation.

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