I am using murine tissue derived cells (mostly lung cells following repeated allergen challenge with HDM) to assess the presence of Th2 cells (CD3+CD4+CD44+IL5 and/or IL13+) and Th17cells (CD4+CD4+CD44+IL17A+). I therefore restimulate these cells ex vivo with PMA/ionomycin in the presence of Brefeldin A for 4h.
Surface staining for the IL33 receptor (ST2) was poor on Th2 cells using antibodies from either eBioscience (RMST2-33; PerCPefFluor710) or BioLegend (DIH9; PerCP5.5 or PECy7). I was wondering if PMA/ionomycin stimulation and/or BrefeldinA would lead to ST2 down-regulation.
We have had good luck with antibodies from R+D or M+D bioscience at 1:100-1:200 dilution. We have not noticed that stimulation of cells for 4h changes ST2 expression levels
For human CD4 T cells from peripheral blood monuclear cells
PMA/Ionomycine does not down regulate; However
the polyclonal PE-goat-anti-human ST2 (somewhat cross-reactive with mouse)
stains well at a 1:10 dilution!
I have recently done an experiment with cells from ST2 deficient mice and looked at cytokines released by lung cells. In the absence of any stimulation (no allergen or PMA/Ionomycin) wild type cells released some IL-5 (likely from ILC2). This is dependent on dying cells releasing IL-33, as no release of IL-5 was detected in ST2 deficient lung cells (ILC2). Thus if your culture conditions (health of cells, stimulation strength...) generate a significant amount of cell death, IL-33 will be released, bind to ST2 and downregulate the receptor)