Good Clone for Delineating FoxP3+ Tregs

February 19, 2019

Immunology
Columbia University
Graduate Student

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Company:

Invitrogen

Product Name:

FOXP3 Monoclonal Antibody (FJK-16s), FITC

Catalog Number:

11-5773-82

We wanted to look at differences in the tumors and tumor draining lymph nodes of mice bearing implanted tumors in terms of lymphocyte infiltration. One of these populations of particular interest to us are FOXP3+ Tregs. This Foxp3 antibody in FITC is decently bright, although it seems to have a decent amount of background over the non-stained sample. We use live CD45+ TCRB+ CD4+ FoxP3+ to identify tregs.

Experimental Design and Results Summary

Applications

Flow Cytometry

Sample

Mouse Tumor Draining Lymph Node

Primary Incubation

30 minutes (Intracellular Stain), RT

Blocking Agent

N/A

Secondary Incubation

N/A

Tertiary Incubation

N/A

Detection

FITC

Results Summary

The tumor draining lymph node was identified as the one closest to the tumor site. It was removed along with the tumor and manually dissociated, followed by centrifugation for fat removal. Cells were stained with surface stains to identify lymphocyte populations for 20 minutes at room temperature, followed by a wash in PBS and then fixation/permeabilization for 45 minutes. Afterwards, intracellular staining for FoxP3 (1:100) as well as other cytokines was performed for 30 minutes at RT in perm buffer. Cells were then washed once in buffer prior to resuspension in PBS for storage at 4°C until cells could be run on the cytometer, about 6 days later on a BD FacsCelesta. The stain was still bright, and gates were set based on a spleen control. Analysis was done on FlowJo software.

DOI or PMID #

N/A

Additional Notes

Must use FoxP3/Transcription Factor fix/perm reagent

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Summary

The Good

Can identify FoxP3+ tregs in tumor DLN

The Bad

Requires special fix/perm, need good positive control

The Bottom Line

Based on the spleen, negative gates were set, and a population of FoxP3+ treg cells could be seen in the TDLN. However, at 1:100, this reagent gets pretty pricey. 1:50 can be used for better resolution but doubles the cost of each experiment. Transcription factor fix/perm must be used for this antibody for optimal staining. Additionally, a good positive control is troublesome to obtain, so we usually use the tumor to see if the staining worked.

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