Anti cd45ra fitc

Peripheral blood mononuclear cells (PBMCs) were isolated from blood samples using Ficoll-Hypaque (GE Healthcare, USA) gradient centrifugation. To obtain Th1/2/17 cells, PBMCs were incubated for 30 min in with specific antibodies (FITC-anti-CD45RA, PE-anti-CCR6, Percep-anti-CD3, PE/Cy7-anti-CD4, APC-anti-CXCR3, BV421-anti-CXCR5, BV510-anti-CCR4; all from BD Biosciences) in PBS containing 2% fetal bovine serum. Cells were examined immediately using a BD FACS-CantoII, and data were analyzed using FlowJo software (TreeStar, USA). Cells were classified as follows: Th17 cells, CD3⁺CD4⁺CD45RA^-CXCR5^-CCR6⁺CCR4⁺; Th2 cells, CD3⁺CD4⁺CD45RA^-CXCR5^-CCR6^-CCR4⁺CXCR3^-; and TH1 cells, CD3⁺CD4⁺CD45RA^-CXCR5^-CCR6^-CCR4^-CXCR3⁺.

Total PBMCs cells were stained with Pacific Blue anti-CD3 (BD 558117, clone UCHT1), PerCP-anti-CD4 (BD 566924, clone SK3), APC-Cy7-anti-CD8 (BD 557834, clone SK1), FITC-anti-CD45RA (BD 555488, clone HI100), and PE-anti-CD45RO (BD 555493, clone UCHL1) antibodies or APC-Cy7-anti-CD19 (BioLegend 302218, clone HIB19), Pacific Blue-anti-CD27 (BioLegend 356414, clone M-T271), FITC-anti-IgD (Life Technology H15501), and PerCP-anti-CD3 (BioLegend 300326, clone HIT3a). After 30 minutes of incubation, cells were washed, fixed, and permeabilized using the Foxp3/transcription factor staining buffer set (Thermo Fisher Scientific) according to the manufacturer’s instruction. Cells were then intracellularly stained with an Alexa Fluor 647-anti-AIOLOS antibody (BD 565215, clone S50-895) for an hour. Cells were acquired and analyzed by flow cytometry (Becton Dickinson FACSCanto II) and FlowJo software (FlowJo 10.8.2, TreeStar), respectively.

Human peripheral blood mononuclear cells (PBMCs) were isolated from freshly drawn venous blood and BM mononuclear cells (BMMCs) were obtained from BM in both of OA and RA patients by Ficoll-Paque PLUS (GE Healthcare Biosciences AB, Uppsala, Sweden) density centrifugation. To examine the phenotype of regulatory T cells (Tregs), the following antibodies were used: anti-CD4-PerCP, anti-CD25-FITC, anti-CD127-PE, anti-CD45RA-FITC, anti-CD45RO-PE and anti-CD184 (CXCR4)-PE (all from BD Biosciences, San Jose, CA, USA). Next, cells were fixed and permeabilized using the Foxp3 Staining Buffer Set followed by intracellular staining with anti-Foxp3-APC antibody (PCH101; eBioscience, San Diego, CA, USA). Cells were acquired using FACSCalibur (BD Biosciences), and the results were analyzed using CellQuest (BD Biosciences) software. The gating strategy was based on the identification of lymphocytes according to FSC and SSC signal distribution. Then, CD4⁺ lymphocytes were gated, and next, appropriate cell populations were identified. Gates were settled according to the isotype control or fluorescence minus one (FMO) for the desired marker. The dot plots shown are representative for at least six experiments done on different patients. The described gating strategy is shown in Figure 2b and was used though all the analysis of the cell phenotype presented in this paper.

PBMCs and spleen MNCs from HLA-A*02:01⁺/B*27:05⁺ and HLA-A*02:01⁺/B*27:05^- individuals were tetramer-stained for 1 hour at room temperature before undergoing dual tetramer-associated magnetic enrichment (TAME), as described [32 (link)], using the MACS PE- and APC-MicroBeads and LS columns (Milteny Biotec, Bergisch Galdbach Germany). Lymphocytes were surfaced stained with the above surface antibodies plus anti-CCR7 PECy7 (BD Pharmingen #557648) and anti-CD45RA FITC (BD Pharmingen #555488). Samples were acquired on a BD Fortessa or BD FACS Aria III for single-cell sorting and subsequent multiplex-nested RT-PCR for TCR analyses of paired CDR3α and CDR3β regions [32 (link)].