Cd31 apc clone ac128

For analysis of Ki67 expression in T-cell subsets, we thawed cryopreserved PBMC and stained them with CD3ε-Alexa700 (clone UCHT1), CD4-APC-H7 (clone SK3), CCR7-BV421 (clone 150503), CD27-BV786 (clone L128), CD49F-BV650 (clone GoH3), CD57-FITC (clone NK-1), CD14-V500 (clone MΦP9), CD19-V500 (clone HIB19) (all from BD Biosciences), CD127-PE-Cy7 (clone R34.34, Beckman Coulter), CD8α-BV570 (clone RPA-T8, BioLegend), CD95-PE (clone DX2, BioLegend), CD45RA-PE-Texas Red (clone MEM-56, ThermoFisher), CD31-APC (clone AC128, Miltenyi Biotec), and LIVE/DEAD fixable Aqua dead cell stain (ThermoFisher) in PBS containing 2% fetal bovine serum (FBS) and 2 mM EDTA. After washing, the PBMCs were fixed with fixation/permeabilization buffer (Transcription Factor Staining Buffer, Ebioscience) for 30 minutes at 4 °C, washed twice with permeabilization buffer (Transcription Factor Staining Buffer, Ebioscience), and stained for 30 minutes with anti-Ki67-FITC (clone 35/Ki-67, BD Biosciences) diluted in permeabilization buffer. Data were acquired on a BD Biosciences LSR Fortessa instrument equipped with 405-nm, 488-nm, 561-nm, and 647-nm lasers and analyzed using FlowJo software (v9.7.8). Naive CD4⁺ and CD8⁺ T cells were defined as CCR7⁺CD45RA⁺ CD95⁻ cells, and Tscm cells were defined as CCR7⁺CD45RA⁺CD95⁺ cells, among CD3⁺CD4⁺ or CD3⁺CD8⁺ live, CD14⁻CD19⁻ lymphocytes.

Enriched populations of cells obtained after magnetic selection protocols were incubated with a cocktail of antibodies and sorted by FACS: CD3ε-FITC (clone UCHT1, BD Biosciences), CD4-APC Cy7 (clone RPA-T4, BD Biosciences), CD8α-BV768 (clone RPA-T8, BD Biosciences), CD45RA-PE-CF594 (clone HI100, BD Biosciences), CCR7-BV450 (clone 150503, BD Biosciences), and CD31-APC (clone AC128, Miltenyi Biotec). Cells were labeled in MACS buffer containing antibodies at 4 °C for 30 minutes and washed extensively and filtered to remove dead cells and debris prior to sorting. FACS was performed using a BD Influx cell sorter equipped with blue (480 nm), red (640 nm), and violet (405 nm) lasers. Sort purities were determined after sorting for all populations.