Anti human cd45ra apc

Peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll-Hypaque density gradient centrifugation (Pharmacia LKB Biotechnology, Piscataway, NJ). Naïve T cells from PBMCs were isolated using a Miltenyi Biotec (Alburn, CA) negative selection kit. Purified naïve CD4+ T cells were activated with plate-bound anti-CD3 (5 μg/ml, BD Bioscience, San Jose CA), soluble anti-human CD28 (1 μg/ml, BD Bioscience), and IL-2 (20 ng/ml, R&D Systems) with or without FTY720 (100 ng/ml, Novartis). After 6 days, cell-free culture supernatants were collected for cytokine analysis by Luminex assay (Miltenyi Biotec), and cells were harvested for RNA extraction and intracellular staining. Naïve T cells were stimulated with PMA (Sigma), ionomycin (Sigma), and Golgistop for 4 h. Cells were stained for anti-human CD4 APC (BD Bioscience) and violet fluorescent reactive dye VVD (Life Technologies), and then cells were fixed and permeabilized with BD fixation and permeabilization buffer and stained for IFN-γ FITC and GZMB FITC (BD Bioscience). For surface staining, the following antibodies were used: anti-human CD4 pacific blue, anti-human CCR7 PE, and anti-human CD45RA APC, IgG2a PE isotype control, IgG2b, and k APC isotype control (all from BD Bioscience). All antibodies were titrated for flow cytometry, which was performed on a BD LSR II (BD Bioscience) and analyzed using Flowjo software.

PBMCs isolated by Ficoll-Paque gradient centrifugation from mutation carrier and sex-matched controls were thawed and plated 3 x 10⁶/ml in RPMI 1640 medium. The cells were allowed to rest for 2 hours and thereafter live and dead cells were stained with LIVE/DEAD™ Fixable Near-IR Dead Cell Stain Kit (Invitrogen; L10119). The cells were washed twice in PBS (Sigma Aldrich; D1408) with 2% FBS and centrifuged at 550 x g for 2 minutes. The cells were stained for surface markers in Brilliant Stain Buffer (BD Biosciences; 563794) for 30 minutes at 4°C, washed, fixed and permeabilized with Transcription Factor Staining Buffer Set (Miltenyi Biotec, 130-122-981) for 30 minutes at 4°C. After permeabilization the cells were washed and stained for FOXP3 for 35 minutes at 4°C. The antibodies against anti-human CD4 BV510 (562970), anti-human CD127 PE Cy7 (560822), anti-human CD45RA APC (561210), anti-human CD25 BV421 (BD Biosciences; 564033), anti-human CCR7 PE (566741), anti-human CCR6 PE (551773) and anti-human CXCR3 PE (550633) were purchased from BD Biosciences. The Alexa Fluor 488 conjugated antibody against human FOXP3 was purchased from Biolegend (Biolegend; 320112) The data was collected with BD LSRFortessa™ using BD FACSDiva software and analyzed with FlowJo™ 10 (BD Biosciences).