Pstat5 antibody

Our hypothesis was tested using IL-15 inducible lung-specific (rtTA-CC-10-IL-15) bi-transgenic experimental asthmatic mice. Cells were isolated from the spleen and mediastinal lymph nodes of DOX and non-DOX exposed CC-10-IL-15 bi-transgenic mice in our experimental asthma model. Isolated cells were stained with anti-CD45-FITC, anti-CD4-PE, anti-CD25-PE-Cy7, and anti-Foxp3-APC and examined by flow cytometry. Cells were first stained with anti-CD45- FITC, anti-CD4-PE and anti-CD25-PE-Cy7 for cell surface markers followed by intracellular Foxp3 Staining Set (eBioscience, San Diego, CA) using an anti-Foxp3-APC antibody. Intracellular IL-10 in regulatory T cells was analyzed using an IL-10 Staining Set (eBioscience, San Diego, CA). The samples were analyzed using FACSCalibur flow cytometer (BD Biosciences, San Diego, CA). ^{38 (link)} STAT5 activation was examined in purified regulatory T cells. Regulatory T cells were isolated using the regulatory T cell Isolation Kit (Miltenyi Biotec, San Diego, CA) from the mouse splenocytes. The isolated regulatory T cells were exposed to 100ng/ml rIL-15 for 0, 15, 30, 60, or 120 min and STAT5 phosphorylation (pSTAT5) was examined using a pSTAT5 antibody (BD Biosciences, San Diego, CA) by flow cytometry and western blot analysis as per the protocol described earlier.^{39 (link)}

For SKM-1 competition assays, live cells were washed with FACS buffer and stained with DAPI before running on a FACSCanto (BD Biosciences). For GM-CSF stimulation experiments, cells were starved overnight, incubated with zombie violet for 20mins, washed, and stimulated with varying concentrations of GM-CSF for 15 minutes. Immediately after stimulation, paraformaldehyde was added to a final concentration of 1.6% and cells were fixed for 10 mins. Cells were then washed with PBS and permeabilized using 2mL of ice-cold 95% methanol. After washing off methanol, cells were stored in FACS buffer until analysis. On the day of analysis, cells were stained with pSTAT5 antibody (BD Biosciences) for 15 minutes, washed and run on a FACSCanto. For PDX experiments, cells were resuspended in 50μL FACS buffer with 2μL of both human and mouse FCR blocking antibody and incubated for 10mins. An antibody cocktail comprised of hCD45, mCD45, hCD3, hCD33, and hCD34(BD Biosciences) was added to each tube, incubated for 15 minutes and washed with FACS buffer. Cells were run on an LSRII (BD Biosciences). Data was analyzed in FlowJo(RRID:SCR_008520).