Brightly Labels Foxp3-Expressing T Cells

January 12, 2017

Immunology
Duke University
Predoctoral

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Quality of Results

Ease-of-Optimization

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Company:

Ebioscience

Product Name:

Anti-Mouse/Rat Foxp3 FITC

Catalog Number:

11-5773-82

The goal of this research study was to determine whether B cell depletion by CD20 mAb altered Foxp3-expressing T cell populations in the spleen and lymph nodes.

Experimental Design and Results Summary

Applications

Flow Cytometry

Sample

Mouse Splenocytes

Primary Incubation

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 (TruStain FcX)

Secondary Incubation

Wash cells once in FACS buffer. Stain cell surface molecules with antibody (1:500 dilution, 100 uL per million cells) for 20 minutes on ice. Wash cells once in FACS buffer. Permeabilize cells using Ebioscience's Foxp3/Transcription Factor Buffer Staining Set (cat. 00-5523) according to manufacturer's instructions.

Tertiary Incubation

Stain with isotype control or Foxp3 antibody (1:100 dilution, 100 uL per million cells) for 30-60 minutes on ice. Wash cells and resuspend cells in 100-250 uL PBS containing 1.5% paraformaldehyde.

Detection

Flow Cytometer

Results Summary

B cell depletion by CD20 mAb does not alter the proportion of Foxp3+ regulatory CD4+ T cells in the spleen of wild-type mice.

DOI or PMID #

10.4049/jimmunol.1302848

Additional Notes

N/A

Related Categories

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Summary

The Good

Foxp3-expressing cells are clearly distinguished using this antibody.

The Bad

Must be used in conjunction with Ebioscience's Foxp3/Transcription Factor Buffer Staining Set

The Bottom Line

To stain Foxp3 requires both the antibody and transcription factor buffer staining set from Ebioscience, but the staining is very robust and repeatable.

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