Fix and perm reagent a

Cryopreserved T-cell subsets from each patient were assessed for levels of pSTAT5 protein by multiparametric flow cytometry as previously described.19 (link) Briefly, cells were resuspended in 100 μL RPMI-1640 supplemented with 10% FBS, fixed with Fix and Perm Reagent A (Invitrogen) for 2–3 minutes at room temperature, and then incubated for 10 minutes with 3 mL cold methanol. Cells were washed in flow buffer (PBS supplemented with 5% FBS) and incubated in 100 μL of Fix and Perm Reagent B (Invitrogen) containing rabbit anti-human pSTAT5 antibody conjugated to alexafluor488, and APC-conjugated extracellular antibodies against CD4 (Beckman Coulter) or CD8 (Beckman Coulter) for 1 hour at room temperature. Fluorochrome-conjugated isotype control antibodies were used to account for background staining. Cells were then washed in flow buffer, fixed in 1% formalin, and analyzed on a Becton Dickinson FACSCalibur flow cytometer (BD Biosciences) using at least 10,000 events gated in the region of the lymphocyte population, as determined by light scatter properties.

PBMCs were isolated from 3 mL of blood by ficoll density gradient centrifugation and adjusted to a density of 2 × 10⁶/mL in Dulbecco's modified Eagle's medium (DMEM; Invitrogen, Carlsbad, CA). Two milliliters of PBMCs were seeded on to a 6-well plate and maintained in DMEM supplemented with 10% fetal bovine serum (FBS; Gibco BRL, Grand Island, NY), 25 ng/mL phorbol myristate acetate, 1 μg/mL ionomycin, and 1.7 μg/mL monensin (all from eBioscience, San Diego, CA), and incubated at 37 °C and 5% CO₂ conditions for 5 hours. Then the cells were collected and incubated with monoclonal antibodies CD3-PC5 and CD8-ECD (Beckman Coulter Immunotech, Brea, CA) for 15 minutes. The cells were permeabilized with FIX and PERM reagent A (Invitrogen, Carlsbad, CA) for 15 minutes, and FIX and PERM reagent B (Invitrogen, Carlsbad, CA) for 10 minutes. Then the cells were incubated with anti-IL-17A-FITC and anti-IL-9A-PE (eBioscience, San Diego, CA) for 20 minutes. After washing with phosphate buffer saline, the phenotypes of the stained cells were analyzed by flow cytometry (COULTER Epics XL, Beckman, Brea, CA). Cells with CD3⁺CD8^– markers were recognized as CD4⁺ T cells and cells with IL-9⁺IL-17^– markers among the T cells were recognized as Th9 cells. The number of CD3⁺CD8^–IL-9⁺IL-17^– cells divided by the total number of CD3⁺CD8^– cells indicated the percentage of Th9 cells in CD4⁺ cells.