Enrichment of CD8⁺ T_CM from PBMCs with clinical grade reagents. (A) Schematic of 2-step immunomagnetic bead enrichment of CD8⁺ T_CM. CD4⁺/14⁺/45RA⁺ cells were removed from PBMCs by negative selection with directly conjugated immunomagnetic beads and CD62L⁺ cells were then positively selected from the remaining population with a biotinylated anti-CD62L mAb and anti-biotin beads. (B) Representative flow plots of PBMC before selection showing the frequencies of CD4⁺, CD14⁺, CD45RA⁺, CD8⁺, and CD62L⁺ cells. FSc indicates forward scatter; and SSc, side scatter. (C) Representative flow plots of the intermediate CD4/14/45RA⁻ fraction showing removal (> 98%) of the depleted subsets. (D) Representative flow plots of the CD62L⁻ and CD62L⁺ fractions after the CD62L selection step. The left 2 panels show the phenotype of the CD62L⁻ fraction and the right 3 panels show the phenotype of the CD62L⁺ fraction. Analysis of the CD8⁻CD62L⁺ cells (far right panel) revealed that the most of these cells were CD13⁺ consistent with the selection of a CD62L⁺ myeloid subset that was not removed with the CD4/CD14/CD45RA depletion. (E) Phenotype of the CD8⁺CD62L⁺ fraction for markers of T_CM. Data are representative of 5 independent experiments. (F) Summary of enrichment data from independent experiments using PBMCs from 5 different donors showing the total frequency of CD8⁺ T cells and the frequency of CD8⁺ cells that are CD62L⁺. The horizontal line indicates the mean ± SEM.