Vioblue conjugated anti cd3

TAng quantification was analyzed by direct flow cytometry following a method previously described [24 (link)]. Briefly, cells obtained from 200 µL of peripheral blood were labelled with VioBlue-conjugated anti-CD3 (Miltenyi Biotec, Madrid, Spain), APC-conjugated anti-CD184 (Miltenyi Biotec, Madrid, Spain) and PE-conjugated anti-CD31 (Miltenyi Biotec, Madrid, Spain) monoclonal antibodies. In a further step, incubation with FACS lysing solution (BD Bioscience, San Jose, CA, USA) was performed to lyse red blood cells. After obtaining the white cell pellets, two washes with PBS were carried out. Finally, a CytoFLEX flow cytometer (Beckman Coulter, Brea, CA, USA) and the Cytexpert 2.3 analyzer (Beckman Coulter, Brea, CA, USA) were used to assess the labeled cells, acquiring approximately 3 × 10⁴ events per sample. CD3⁺ cells were gated and then assayed for the expression of CD184 and CD31 in the lymphocyte gate. TAng were considered as triple-positive for CD3, CD184 and CD31 (Figure S1) and expressed as percentage of cells in the lymphocyte gate.
EPC and EC frequencies were measured by flow cytometry following the method previously described [14 (link),15 (link)]. EPC were considered as CD34⁺, CD45^low, CD133⁺ and CD309⁺ cells and EC were defined as triple-negative for CD34, CD45 and CD133 and positive for CD309, following the nomenclature previously defined [14 (link),15 (link)].

Percentages of CD3+CD4+CD28-null T cells (senescent T helpers) and CD3+CD8+CD28-null T cells (senescent T cytotoxics) were determined by flow-cytometric analysis. PBMC were stained with VioBlue conjugated anti-CD3, Viogreen conjugated anti-CD8, PE-VIO 770A conjugated anti-CD4 and APC-VIO 770A anti-CD28 antibodies (Miltenyi Biotec). Cells were analyzed with a MACSQuant Flow Cytometer (Miltenyi Biotec). The percentage of CD28null T cells within the CD4+ or CD8+ T cell population was then calculated.