Anti mouse RorgT PE to Quantify Rorgt+ Expressing Cells Including ILC3, Th17 and Rorg+ Tregs

NOMIS CENTER FOR IMMUNOBIOLOGY AND MICROBIAL PATHOGENESIS
The Salk Institute of Biological Studies
Postdoctoral Researcher

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Quality of Results

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Company:

Invitrogen

Product Name:

Anti mouse RorgT PE

Catalog Number:

12-6988-82

Clone Number:

AFKJS-9

I study how intestinal immune system matures during breastfeeding phase under the influence of maternal factors. I routinely analyze different immune cell population in mouse gut. Recently I analysed different T regs subset and I used this Anti mouse RorgT PE from Invitrogen to quantify the RorgT + Tregs in mouse gut during postnatal breastfeeding phase.

Experimental Design and Results Summary

Applications

Flow Cytometry

Sample

Mouse immune cells from any tissue

Primary Incubation

1:400

Blocking Agent

Not needed

Secondary Incubation

Not needed

Tertiary Incubation

Not needed

Detection

Any flow cytometer with 561 nm laser

Results Summary

A total of 5 × 10⁶ immune cells were isolated from the small intestine of postnatal day 14 (P14) mice and stained with the fixable viability dye Zombie NIR for 20 minutes on ice. Following viability staining, cells were incubated with fluorochrome-conjugated antibodies against CD45, TCRβ, and CD4 (all at a 1:800 dilution) for 30 minutes on ice. Cells were then fixed and permeabilized using the Invitrogen Foxp3/Transcription Factor Staining Buffer Set according to the manufacturer's protocol, with fixation performed for 30 minutes on ice. After washing with permeabilization buffer, intracellular staining was performed using antibodies against RORγt (PE), Foxp3, and Helios in permeabilization buffer at 4 C overnight. RORγt⁺ regulatory T cells were identified by gating sequentially on live CD45⁺ TCRβ⁺ CD4⁺ Foxp3⁺ cells, followed by analysis of RORγt expression.

Additional Notes

If you are doing an overnight staining use it at lower dilution. In my hand it works on 1:400 dilution in 200ul staining volume

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Summary

The Good

Very good antibody, you should see a distinct RorgT treg population if you are following above protocol.

The Bad

Nothing bad about this. Works great

The Bottom Line

Cheap and great antibody, will keep buying it!

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