Phospho-S6 PE For FACS

June 05, 2017

Microbiology & Immunology
Columbia University
MD/PhD Student

Overall

Quality of Results

Ease-of-Optimization

What do these ratings mean?
Write a Review

Company:

Cell Signaling Technology

Product Name:

Phospho-S6 Ribosomal Protein (Ser235/236) (D57.2.2E) XP® Rabbit mAb (PE Conjugate)

Catalog Number:

5316S

TCR stimulation of CD4+ T cells induces metabolic reprogramming to facilitate proliferation and acquisition of effector functions. Our lab is interested in the role of the mTOR kinase during CD4+ T cell proliferation and how this activity is linked to cell fate. In in vitro experiments, CD4+ T cells were activated with anti-CD3/CD28 antibodies and subsequently analyzed by phospho-Flow for transcription factor expression and mTOR activation with phosphorylation of the ribosomal subunit S6 as a proxy. mTOR activity was assessed in the context of metabolic inhibitors as well as activators of T cell differentiation.

Experimental Design and Results Summary

Applications

Flow Cytometry

Sample

Purified mouse CD4+ T cells

Primary Incubation

1:200, 45 minutes, 4C

Blocking Agent

1X eBioscience Perm/Wash buffer

Secondary Incubation

N/A

Tertiary Incubation

N/A

Detection

BD Fortessa

Results Summary

CD4+ T cells were activated for 3 days with CD3/CD28 stimulation and supplemental IL-2, followed phospho-flow analysis for pS6 as a marker of mTOR activity. Cells were stained following a two-step paraformaldehyde/methanol fixation protocol which was found to be essential for efficient detection of phosphorylated antigens. First, cells were fixed with 4% paraformaldehyde for 15 minutes at room temperature, washed twice with dPBS, followed by subsequent fixation/permeablization with ice-cold 100% methanol for 5 minutes at -20C. With this method, strong pS6 signal was detected in activated CD4+ T cells. A portion of activated cells, but not all, exhibited high levels of mTOR activity, as evidenced by pS6 signal.

DOI or PMID #

N/A

Additional Notes

No signal detected if fix/perm is done only with Foxp3 transcription factor kit.

Related Categories

Image Gallery

Summary

The Good

Strong pS6 signal and clear separation

The Bad

Requires two-step paraformaldehyde/methanol fixation as with other phospho-epitopes

The Bottom Line

The pS6-PE antibody conjugate allows for strong, efficient detection of mTOR activity in cultured lymphocytes.

Share your experience with other scientists. Write a Review! »

Join the discussion