Does anyone have a good protocol for flow cytometry on the brain looking at T cell profiles? Specifically Tregs?
For flow cytometric analyses you can use a wide variety of reagents and antibodies. I recommend the following among many that various papers with similar protocols use for Tregs: fluorescein isothiocyanate (FITC), phycoerythrin (PE), peridinin chlorophyll protein-cyanine 5.5 (PerCP-Cy5.5), allophycocyanin (APC).
Also, use Viability Dye eFluor 780 (FVD780) to remove dead cells.
For isolation of brain Tregs, cut or mince brains and enzymatically dissociate them with 2 mg ml−1 collagenase D (Roche Applied Science), and 2 mg ml−1 DNase I in RPMI-1640 for 45 min at 37 °C. Centrifuge the tissue and resuspend in 30% Percoll (GE Healthcare Life Science) layered on 70% Percoll, and centrifuge at 2,000 r.p.m. for 20 min. Cells can be collected from the 70–30% interface. Isolated cells can be stained with fluorochrome-conjugated antibody and sorted by, for instance FACSAria IIu (Becton Dickinson) or can analysed by FACSCanto II (Becton Dickinson) and FlowJo software (Tree Star). Then you can assess the efficiency of brain Treg cell depletion by flow cytometry.
See the following for a similar detailed methods:
https://www.nature.com/articles/s41586-018-0824-5?WT.feed_name=subjects_t-cells
I just found another similar protocol for flow cytometry analysis that can be helpful:
" Cells were stained for FACS using the following antibodies: CD4-PerCP-Cy5.5, CD4 Alexa 647, MHCII-FITC, CD45.2-APC, CD11c-PE, CD3-PE-Cy7, CD8a-APC, CD69-PerCPCy5.5, IL-10-PE, CD11b-APC-Cy7, Ly6G-FITC (BD Biosciences) and Foxp3-PE or FITC, IFN-γ-PE, TNF-α-PE, Ki-67 Alexa 700 from eBioscience. For intracellular staining, cells were restimulated with 100ng/mL phorbol myristate acetate (Sigma-Aldrich) and 600ng/mL ionomycin (Sigma-Aldrich) in the presence of protein transport inhibitor (BD GolgiStop or GolgiPlug, BD Biosciences) in RPMI containing 10% fetal calf serum for 4 hours and stained with CD3, CD45, CD4, IL-10, CD11b, IFN-γ-PE, TNF-α and Foxp3 according to the manufacturer’s instructions (eBioscience). Data were collected on a LSRII flow cytometer (Becton Dickinson, Heidelberg, Germany) and analyzed by FlowJo software (Tree Star, Ashland, OR)."
Check their paper for further details:
Article Amplification of Regulatory T Cells Using a CD28 Superagonis...
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