Cd4 t cells isolation kit

For IRBP_1-20-specific T cell assay, the IRBP_1-20-induced EAU mice were injected with cysteamine (40 mg/kg) intraperitoneally for 14 days. After the splenocytes were isolated from the IRBP_1-20-injected mice treated or untreated with cysteamine, they were re-stimulated with IRBP_1-20 (1 ug/ml) in vitro. After 24 hr, the supernatants from the cultures media were collected and assayed by ELISA for the detection of IL-22 levels. PBMCs were isolated from the blood samples of uveitis patients (n = 6) and healthy donors (n = 6). Using the CD4+ T cells isolation kit (130-096-533, Miltenyi Biotec, Auburn, CA, USA), human CD4+ T helper cells are isolated from PBMCs. The CD4+ T cells were cultured in the presence or absence of IRBP_1-20 (1 μg/ml) and cysteamine (2.5 μg/ml) in vitro for 24 h. The culture supernatants were collected and used for the detection of IL-22 via ELISA.

CD4⁺ T cells from the spleen of FoxP3(GFP) mice were negatively selected using a CD4⁺ T cells isolation kit (Miltenyi, Auburn, CA, USA) and were activated with αCD3-coupled microbeads in a round bottom 96-well plate in the presence or absence of Fc-GITR-L (1 μg/ml) for 2 days as described previously (9 (link)). Cells were stained with CD4 and CD103. Expression of FoxP3 was judged by the reporter protein EGFP. Cell numbers were counted with a Countess Automated Cell Counter (Invitrogen, Grand Island, NY, USA).

Heparinized peripheral blood obtained from patients was layered onto a Ficoll-Histopaque 1077 (Sigma-Aldrich, St. Louis, MO, USA) density gradient and centrifuged at 2,000 rpm for 30 minutes. Mononuclear cells were collected at the interfaces, washed and re-suspended in Phosphate Buffer Saline (PBS), supplemented with 2mM EDTA, and further enriched with CD4+ lymphocytes by negative selection using an appropriate CD4+ T cells isolation kit (Miltenyi Biotec, Bergish Gladbach, Germany).
For Tregs evaluation, CD4+ lymphocytes were stained with an Antibody cocktail containing CD4+ PerCP Cy5.5, CD25 PE and CD127 FITC (all from BD Biosciences Pharmingen, San Diego, CA, USA), for 20 minutes at room temperature, and subsequently fixed and permeabilized for intranuclear FoxP3 expression with an anti-human Foxp3-APC staining set (eBioscience, San Diego, CA, USA), accordingly to manifacturer’s instructions. Acquisitions were made on BD-FACSCanto II Flow Cytometer, equipped with FACS Diva Software; Treg cells were identified in the CD4+ CD25 high, CD127^_ Foxp3+ cells fraction and expressed as percentage of total CD4+ lymphocytes.