Pe conjugated anti il 17a antibody

Spleens were ground and filtered through a 70 μm cell strainer. RBCs were lysed using RBC lysis buffer (eBioscience). Isolation of renal leukocytes was processed as discussed in the previous step. Approximately 1x10⁶ of either spleen of kidney cells were resuspended in RPMI medium supplemented with 5% FBS and stimulated with 2 µl of BD Leukocyte Activation Cocktail (ionomycin and phorbol 12-myristate 13-acetate (PMA) along with the Golgi inhibitor, brefeldin A) at 37°C for 5 hrs. Cells were washed and stained first with LIVE/DEAD® Fixable Violet dead cell stain (Invitrogen, Carlsbad, CA). The following surface antibodies were then added for 30 minutes: BV510-conjugated anti-CD45 antibody (clone 30-F11), PerCP-Cy5.5-conjugated anti-CD3 antibody (BioLegend, clone 17A2), and APC-H7-conjugated anti-CD4 antibody (BD Biosciences, clone GK1.5). Intracellular staining was then performed with the BD Cytofix/Cytoperm^TM Plus fixation/permeabilization solution kit following the manufacturer's instructions (BD Biosciences) and using phycoerythrin (PE)-conjugated anti-IL-17A antibody (eBioscience, clone eBio17B7). We followed exactly the same protocol to detect RORγt in blood and gastric tissues, however, we did not stimulate with ionomycin/PMA. PE-conjugated anti-RORγt antibody (eBioscience, clone B2D) was used.