Facsaria lsr 2

After isolation, cells were resuspended in phosphate-buffered saline (PBS) containing 2 mM EDTA and spun for the removal of contaminating platelets. Prior to sorting, peripheral CD4⁺ T cells were enriched with the use of magnetic beads and column purification (Miltenyi Biotec). Enriched peripheral CD4⁺ T cells were then stained with previously determined volumes of the following fluorescently conjugated MAbs: CD3-APC-Cy7 or CD3-AF700 (clone SP34-2), CCR7-PE-Cy7 (clone 3D12), CD8-APC-Cy7 (clone SK1), CD45RA-APC (clone 5H9), CD95-PE-Cy5 (clone DX2), and CD62L-PE (clone SK11) from BD Bioscience; CD28-ECD (clone CD28.2) from Beckman Coulter, and CD4-BrilliantViolet650 (clone OKT4) and CD8-BV421 (clone RPA-T8) from BioLegend. Circulating populations for sorting were defined as follows: naive, CD45RA⁺ CCR7⁺ CD95⁻ SCM, CD45RA⁺ CCR7⁺ CD95⁺ CD28⁺ CD62L⁺; CM, CD45RA⁻ CD95⁺ CCR7⁺ CD62L⁺; and EM, CD95⁺ CCR7⁻. Sorting was performed on a FACSAria LSR II (BD Biosciences) equipped with FACSDiva software.

For cell sorting, peripheral CD4⁺ T cells were first enriched by negative selection with the use of magnetic beads and column purification (nonhuman primate CD4⁺ T cell isolation kit; Miltenyi). Enriched CD4⁺ T cells were then stained with viability dye (Live/Dead Aqua) and previously determined volumes of the following fluorescently conjugated MAbs: CD3-AF700 (clone SP34-2), CD8-APC-Cy7 (clone SK1), CD95-PE-Cy5 (clone DX2), CD62L-PE (clone SK11), and CCR7-PE-Cy7 (clone 3D12) from BD Biosciences; CD4-BV650 from BioLegend. Sorted live CD3⁺CD8-CD4⁺ populations were defined as follows: naive cells, CD62L⁺ CCR7⁺ CD95-; and memory, CD95⁺. Sorting was performed on a FACSAria LSR II (BD Biosciences) equipped with FACSDiva software.