Cd34 apc

Cells were washed with PBS and stained at room temperature using a 1:10 dilution of the indicated antibodies in 100 µL PBS for 10 min. Cells were washed once more to remove antibody excess and flow cytometry data was subsequently acquired with FACSCalibur (BD Biosciences, Franklin Lakes, NJ, USA). Live cells were gated based on forward and side scatter plots. Isotype staining’s were performed as control conditions. Anti-CTLA-4 PE extracellular antibody, anti-Gal-9-PE and their corresponding isotype controls were purchased from Biolegend, San Diego, CA, USA. The rest of the antibodies (anti-CD276-PE, anti-CD80-PE, anti-CD86-APC, anti-CD273-PE, anti-CD274-APC, anti-HLA-E-APC, anti-CD112-PE, anti-CD66abce-APC, anti-CD155-PE, anti-CD19-APC or FITC, anti-PD1-PE, anti-TIM3-APC, anti-NKG2A-FITC, CD56-APC, CD96-APC, TIGIT-PE, CD34-APC, CD271-PE and corresponding isotype controls) were obtained from Miltenyi Biotec, Bergisch Gladbach, Germany. CD19-CAR and CD276-CAR expression on NK-92 cells was determined by CD271 and CD34 marker gene expression, respectively, whereas luciferase expression on target cells was correlated with GFP for Nalm-6 GFP/Luc and CD19 marker expression for U-937 CD19tag/Luc.

For flow cytometry analysis, oBMSC were stained with the following anti-human antibodies: CD34-APC, CD90-FITC, CD73-APC, CD105-PE, CD146-APC, CD271-PE, HLA-DR-PE, and isotype controls (Miltenyi Biotec). Additionally, the following anti-sheep antibodies were used: CD45-FITC and CD44-FITC (Bio-Rad). Cells were stained as per the manufacturers’ instructions, and analysis was completed using an LSR II flow cytometer (BD Biosciences). In this analysis, we also included sheep bone marrow mononuclear cells (MNC) and hBMSC. These cell populations served as controls for antibody performance against a common hBMSC population, and for sheep MNC, where haematopoietic cells (CD45⁺) would be expected. Data were analysed using FlowJo software, version 10 (BD Biosciences).

Detached cells were briefly resuspended in 200 µL of FACS buffer (1% FBS in PBS) and incubated with antibodies for 30 min. We used CD29-APC, CD44-APC, CD90-APC, CD105-APC as MPC markers, and CD34-APC, CD45-APC, SSEA4-APC, TRA-1-60-PE (Miltenyi Biotec, Bergisch Gladbach, Germany) as hematopoietic and stem cell markers. Cells were washed twice with FACS buffer and analyzed using a FACSCalibur cytometer (BD Biosciences, San Jose, CA, USA).

Cells were detached with trypsin, fixed with 4% PAF for 10min and then washed twice with PBS. Cells were re-suspended in PBS with 0.5% FBS. Cells were labeled with the following anti-human antibodies: CD105-APC, CD73-APC, CD90-APC, CD44-APC, CD34-APC, and CD31-APC (Miltenyi), CD45-APC (Becton Dickinson) for immunophenotyping assays; CD49a-APC and CD49d-APC (Miltenyi), CD106-APC and CD54-APC (Becton Dickinson) for adherence assays; Rabbit anti-p21, Mouse anti-p27, Mouse anti-Cyclin B1, Rabbit anti-Cyclin D1 (all from Cell Signaling), and Rabbit anti-p19 (Upstate) for cell cycle assays. Donkey anti-Mouse IgG DyLight650 and Donkey anti-Rabbit IgG DyLight650 (1:200 dilution for each, Thermo Scientific) were used as secondary antibodies when needed. Isotype antibodies served as respective controls. For intracellular labeling, cells were permeabilized with PBS/0.1% Triton X100 solution (BioRad). Cells were acquired on a FACS Scan flow cytometry analyzer (FACs Calibur, Becton Dickinson) and analyzed using CellQuestPro software (Becton Dickinson).