Hla dr percp

To characterize monocyte subsets, freshly purified PBMCs were analyzed by six-color flow cytometry on FACS LSRII apparatus. The gating strategy was based on a previous report [15 (link)]. Monocytes were subdivided into three major subsets: classical CD14⁺⁺CD16⁻, intermediate CD14⁺⁺CD16⁺ and non-classical CD14⁺CD16⁺⁺ monocytes. The following anti-human mAbs were used: CD45-Amcyan (BD Biosciences), HLA-DR-PerCP (BD Biosciences), CD19-ECD (Beckman Coulter), CD14-QDot655 (Invitrogen), CD16-APC-H7 (Beckman Coulter, Villepinte, France). The Live/Dead blue Dye (Invitrogen) was used to exclude dead cells.
Samples of the purified monocytes used to generate MD-DCs and of the resulting MD-DCs were routinely stained with the following anti-human mAbs: CD14-FITC, CD11c-APC, CD40-PE, HLA-I-FITC, HLA-DR-PerCP, CD80-PE, CD83-APC and CD86-FITC (all from BD Bioscience) and analyzed by flow cytometry on FACS canto II apparatus (BD Biosciences).

Flow cytometry analyses were performed on passages P1, P5, and P11 with the following fluorescently labeled monoclonal antibodies: FITC-CD73, FITC-CD90, FITC-CD105, PE-CD45, PE-CD34, and PercP-HLA-DR (BD), according to the method described by Maumus et al. (28 (link)). Cells were incubated with monoclonal antibodies for 15 min at room temperature (22±2°C) in the dark and then washed with PBS before fixation with 1% formaldehyde (Sigma-Aldrich). After incubation, cell viability was analyzed using a three-color flow cytometer (FACS Calibur, Becton Dickinson, USA); 10,000 events were analyzed for each sample. For data acquisition and analysis, the Cell Quest and Paint-a-Gate softwares (Becton Dickinson Pharmigen) were used

Bone marrow samples from patients with SAA and HCs were collected in heparin anticoagulant tubes. Bone marrow mononuclear cells were isolated by density gradient centrifugation using Ficoll‐Paque Plus solution (Amersham Biosciences, Uppsala, Sweden) according to the manufacturer's instructions. Thereafter, we placed the cells in a culture medium containing 79% RPMI 1640 (Gibco‐BRL, Grand Island, NY, USA), 20% foetal bovine serum (Gibco‐BRL), and 1% penicillin–streptomycin (Hyclone, South Logan, UT, USA) at a cell concentration of 1.5 × 10⁶/mL. After incubation for 2 h, the suspended cells were discarded, and the remaining semi‐adherent cells were cultured in a medium containing 100 ng/mL recombinant human granulocyte monocyte colony stimulating factor (rhGM‐CSF) (North China Pharmaceutical Co., Hebei, China) and 25 ng/mL recombinant human (rhIL‐4) (PeproTech, Rocky Hill, NJ, USA) at 37°C with 5% CO₂. The medium was changed, and the above‐mentioned cytokines were added every 2 days. On Day 6, 1000 U/mL rhTNF‐α (PeproTech) was added to induce mDC maturation. On Day 7, the suspended cells were collected, labelled with APC‐CD11c (BD Pharmingen) and PerCP‐HLA‐DR (BD Pharmingen), and sorted using a FACSAria flow cytometer (BD Biosciences, San Jose, CA, USA). Purity of the mDCs was measured and quantified as the percentage of CD11c⁺HLA‐DR⁺ cells among all sorted cells.