Cd45 viogreen

PBMCs isolated from 13 obese children (6 male, 7 women) were treated with Ctrl, EVOO and olive oil extracts for 24 h. After the treatment, PBMCs were detached from the plate with cold D-PBS 1X (Gibco, New York, NY, USA) + 0.5 mM EDTA (Thermo Fisher Scientific, Waltham, MA, USA), washed with D-PBS 1X + 2 mM EDTA (Thermo Fisher Scientific, Waltham, MA, USA) + 0.5%BSA (Sigma-Aldrich, St Louis, MO, USA), and stained with CD14 FITC/CD64 PE (Becton, Dickinson and Company, OR, USA, REF:333179), CD16 APC and CD45 VioGreen^® (Miltenyi Biotec, Bergisch Gladbach, Germany, REF:130-113-951 and REF:130-110-776, respectively) according to the manufacturer’s instructions. To test cell viability, 7-AAD PerCP-Cy5.5 (7-Aminoactinomycin D, Becton, Dickinson and Company, OR, USA) was used. Flow cytometer acquisition was performed using the FACSCanto II flow cytometer (Becton, Dickinson and Company, OR, USA), and data were analyzed using Diva software, version 8.0.1 (Becton, Dickinson and Company, OR, USA). Gating strategy: after the selection of single and live cells from PBMCs, cells were checked for positivity to CD64 and CD45 (each marker vs. side scatter). CD64⁺ CD45⁺ cells were analyzed for the expression of CD16 and CD14 by a dot plot (Supplementary Figure 1).

Peripheral blood (PB) from healthy blood donors (HBDs) and from AL patients were obtained after informed consent. The study was approved by the Medical Ethics Committee of the Canton Geneva (study no. 08-001 and 2020-00176). Diagnosis and clinical and laboratory characteristics of leukemia patients are detailed in Table S1. CD34⁺ cells were enriched using the UltraPure CD34 Microbead kit (Miltenyi Biotec, Bergisch Gladbach, Germany, #130-100-453) and magnetic-activated cell-sorting separation columns (Miltenyi Biotec, #130-042-201). CD34⁺ cell viability and purity were assessed by flow cytometry after staining cells with CD34-PE-Vio770 (Miltenyi Biotec, clone: AC136, #130-113-180), CD45-VioGreen (Miltenyi Biotec, clone: REA747, 130-110-638), and 7-Amino-Actinomycin D (7-AAD) viability dye for live/dead cell discrimination (Beckman Coulter, Brea, California, CA, USA, #B88526). Samples were analyzed on a Navios 10-color flow cytometer (Beckman Coulter, Brea, CA, USA) and the data interpreted with the Kaluza software (Analysis Version 2.1, Beckman Coulter). CD34⁺ cells were subsequently cultured in StemSpan^TM SFEM II serum-free medium (STEMCELL Technologies, Vancouver, BC, Canada, #09605), supplemented with Flt3L, IL-3, IL-6, SCF and TPO (StemSpan^TM CD34⁺ expansion supplement; STEMCELL Technologies, #02691).

Anti-VISTA antibodies and isotype control (Human IgG1 Isotype control, Bio X Cell BP0297) were labeled with Alexa Fluor 647 using the protocol in the conjugation kit (Biotium). Monocytes, T-cells, and Neutrophils were labeled in a human blood sample (BIOIVT) by flow cytometry using CD45-VioGreen (130-110-638), CD3-PE-Vio 770 (130-113-140), CD16-Vio Bright B515 (130-119-616) and CD14-PE (130-113-147) in the presence of FcR Blocking Reagent (130-059-901; all reagents from Miltenyi Biotec and antibodies diluted 50-fold as per manufacturers recommendation). Samples with appropriate FMO (Fluorescence Minus One) controls for each antibody were analyzed in parallel. RBC were lysed using Lysis Buffer (BD Biosciences 555899). Samples were washed in 1x PBS pH7.4 containing 1% heat inactivated fetal bovine serum, and propidium iodide (PI) staining (Miltenyi 130-093-233) for Live/dead cell discrimination done immediately before analysis using a MACS Quant Analyzer (Miltenyi). For NK cells, isolated human NK cells (Hemacare/Charles River Labs) were stained with labeled anti-VISTA or isotype control mAbs and CD56-FITC (Miltenyi 130-114-740; 50-fold diluted) in MACSQuant Running Buffer (Miltenyi 130-092-747), and Sytox Blue (Thermo Fisher Scientific S34862) was used for gating for live cells.

For FACS analysis, healthy and lesional skin (both from corresponding anatomical sites) or blood was taken from mice after induction of experimental EBA. Single cell solutions from and blood were erythrocyte lysed with RB cell lysis buffer (Miltenyi). The skin samples were cut into small pieces and digested with 345 mg/ml liberase (Roche) in RPMI for 30 min/37°C. Single cells were stained for the following surface markers using standard FACS procedures: CD45-VioGreen (clone: 30F11), CD3-VioBlue (clone: 17A2), Ly6C-FITC (clone: 1G7.G10), as well as Ly6G-APC Vio770 (clone: 1A8) and for blood CD19-APC (clone: 6D5), all from Miltenyi. For the subsequent intracellular staining, the cells were fixed in fixation buffer (BioLegend) and permeabilized using the Intracellular Staining Perm Wash Buffer (BioLegend) following the manufacturer’s protocol. Intracellular staining was performed with SYK-PE (clone: 4D10.2). Cells were first gated for scatter (SSC-A/FSC-A) and singlets (FSC-H/FSC-A). The CD45⁺ gates were further analyzed for double-positive staining of SYK with the appropriate cell markers. Measurements were performed at the Miltenyi MacsQuant10, and data were analyzed with the MACSQuantify™ Software (version 2.8).