Facsaria 2 cell flow cytometer

The sorting of CD4⁺ T cells or CD8⁺ T cells were labeled with 10 μM carboxyfluorescein succinimidyl ester (CFSE) (Thermo CellTrace™ CFSE Cell Proliferation Kit C34554) for incubation at room temperature in the dark for 10 mins. The cells were centrifuged and then washed three times with Roswell Park Memorial Institute 1640 (RPMI 1640) containing 10% FBS. CD4⁺ or CD8⁺ T cells were seeded in 24-well plates (5 × 10⁵ per well) and co-cultured with magnetic beads (Miltenyi, T-cell activation/expansion kit, 130–093–627) of the same proportion in RPMI 1640 supplemented with 10% FBS and IL-2 (50 U/mL) according to the manufacturer’s protocol. After 48 h, 96 h, and 144 h, CD4⁺ T-cell and CD8⁺ T cells were partially harvested and stained with anti-CD4 (1:100, ab133616, Abcam) or anti-CD8a (1:500, ab217344, Abcam). Proliferation was detected by FACSAria II cell flow cytometer (BD Biosciences, CA, USA). The data were analyzed with FlowJo softwar.

Harvesting the spleens of Ogr1^−/− and wild-type (WT) mice and keep them in PBS, and then gently homogenized the spleens with a syringe plunger in a 72 µM sieve. After that, transfer the grinding fluid to a conical tube with centrifugation for 10 mins at 300 × g, 4°C. Remove the supernatant and resuspend the splenocytes with red blood cell (RBC) lysis (BD Pharm Lyse, 5075567). The sorting of CD4⁺ T-cell and CD8⁺ T cells Using CD4⁺ T-Cell Isolation Kit and CD8⁺ T-Cell Isolation Kit (Miltenyi, 130–104-454, 130–104–075) following manufacturer’s instructions. Cellular purity was detected with FACSAria II cell flow cytometer (BD Biosciences, CA, USA) after staining with anti-CD3 (1:1000, ab16669, Abcam, Cambridge, MA, USA) and anti-CD4 (1:100, ab133616, Abcam) or anti-CD8a (1:500, ab217344, Abcam) antibody. The data were analyzed with FlowJo softwar. Isolated T cells were cultured in RPM-1640 supplemented with 10% fetal bovine serum (FBS). When adjusting the acidic medium in vitro, we first added 20 mm MOPS (3-(n-morpholinyl) propane sulfonic acid) to the medium containing FBS to minimize the pH change during cell culture and then adjusted the pH value to ph6.6 with 1 N HCl [12 (link), 13 (link)]. After 24 hours, the stability of pH value was evaluated by pH meter.