Cd25 percp cy5

Fc-gamma receptors were blocked with a rat anti-mouse CD16/CD32 antibody (BD). Mouse-specific antibodies were the following: CD3-Brilliant violet 510, CD25-PercP Cy5.5, I-A/I-E-Brilliant violet 510, CD45R/B220-PE-Cy7, CD43-APC, CTLA-4-APC (all from BioLegend), CD4-APC-Cy7, CD69-PE, CD11c-PercP Cy5.5, CD19-APC-Cy7, CD40-FITC (all from BD Pharmingen), CD8α-PE-Cy7, FoxP3-FITC, CD86-APC, CD83-FITC, CD80-PE-Cy7, IgM-PercP-eFluor 710 (all from eBioscience).
During the experimental set-up, CD3 antibody was included in the staining panel (BV510 BioLegend clone 17A2) and used for the gating of T cells. The results obtained with gating on CD3+ CD4+ CD8- were similar to those obtained with gating on CD4+ CD8-. Due to the limitation in the maximum number of colours that can be discriminated with the FACS Canto II, CD3 staining was omitted in subsequent stainings.
Dead cells were excluded using the fixable viability dye-eFluor 450 (eBioscience), and intracellular staining was performed using the fixation/ permeabilization buffer set from eBioscience. Flow cytometry measurements were performed on a FACS Canto II instrument (BD) and the data were analyzed with FlowJo software (Tree Star).

Fc‐gamma receptors were blocked by incubating human cells with 200 µg/mL of IVIg (Octagam; Octapharma), and mouse samples were incubated with a rat anti‐mouse CD16/CD32 antibody (BD clone 2.4G2) diluted 1:100. Human samples were stained with the following antibodies (final concentration in the well is indicated for each antibody): CD3‐Brilliant Violet 510 1:100 (BioLegend, San Diego, CA, USA, clone OKT3), CD4‐APC‐CY7 1:100 (BD Pharmingen clone RPA‐T4), CD25‐APC 1:100 (BD Pharmingen clone 2A3) Foxp3‐PE 1:50 (eBioscience, San Diego, CA, USA clone 236A/E7). Mouse‐specific antibodies were the following: CD3‐Brilliant Violet 510 1:50 (BioLegend clone 17A2), CD4‐APC‐CY7 1:100 (BD Pharmingen clone GK1.5), CD8α‐PE‐Cy7 1:100 (eBioscience clone 53‐6.7), CD25‐PercP Cy5.5 1:100 (BioLegend clone 3C7), FoxP3‐FITC 1:50 (eBioscience clone FJK‐16s). Dead cells were excluded using the fixable viability dye‐eFluor 450 1:1000 (eBioscience), and intracellular staining was performed using the fixation/permeabilization buffer set from eBioscience). Flow cytometry measurements were performed on a FACS Canto II instrument (BD) and the data were analyzed with FlowJo software (Tree Star).

Release tests for MNC-QQ were conducted to evaluate potency based on the ratio of cells and their vasculogenic potential. The presence of cell types essential for vascular and tissue regeneration in the MNC-QQ culture was confirmed using flow cytometry antibody-based detection of the following cell surface markers: CD34-PE, CD206-PE/Cy7, CD192(CCR2)-PerCP/Cy5.5, CD3-Alexa700, CD14-APC/Cy7, CD4-FITC, CD8-APC/Cy7, CD31-APC/Cy7, CD19-FITC, CD56-BV421, CD25-PerCP/Cy5.5, CD127-BV421 (BioLegend, San Diego, CA, USA), CD133-APC (Miltenyi Biotec, San Diego, CA, USA), and CXCR4-APC (BD Biosciences).^{17 (link),23 (link)}The vasculogenic potential of MNC-QQ was evaluated using a MethoCult SF H4236 colony formation assay (STEMCELL Technologies, Vancouver, BC, Canada).^{22 (link)} Cells (2 × 10⁵ per dish) were inoculated into a 35-mm Primaria culture dish and cultured for 14 days under routine conditions. Clusters comprising more than 100 cells were counted as primitive EPC CFU or definitive CFU based on the cell size and shape under phase-contrast microscopy (Nikon, Tokyo, Japan). PB collected less than 1 month before cell therapy was used to compare the baseline vasculogenic potential and cell surface markers between PBMNCs and MNC-QQ cells harvested on the treatment date.

Regulatory T cells (Tregs) were identified by surface staining with anti-CD3 Alexa700 (Exbio, Vestec, Czech Republic), CD4 PC7 (eBioscience, San Diego, CA), CD8 PE-Dy590 (Exbio), CD25 PerCPCy5.5 and CD127 Alexa647 (BioLegend, San Diego, CA) antibodies, followed by fixation and permeabilization with a FoxP3 staining buffer set (eBioscience) and intracellular staining with anti-FoxP3 FITC (eBioscience), as previously described [27 , 28 ]. All samples were processed and analyzed immediately after blood sampling on FACSAria™ (Becton Dickinson, Heidelberg, Germany) and analyzed using FlowJo software (Tree Star, Ashland, OR).