High-purity 4-way sorting of a 12-color immunophenotyping panel
Whole blood was lysed with RBC Lysis Buffer and stained with Alexa Fluor® 488 CD3, PerCP-Cy™5.5 CD4, BD Horizon Brilliant™ Violet 570 (BV570) CD8, APC-Cy™7 CD14, PE/Dazzle™ 594 CD16, BV421 CD19, BV605 CD20, BV510 CD27, APC CD45RA, PE-Cy7 CD56, Alexa Fluor® 700 HLA-DR, and PE TCR gamma/delta antibodies. Cells were incubated for 20 minutes on ice, washed 2X with staining buffer, and analyzed on the MA900 equipped with 488-nm, 405-nm, and 638-nm lasers.
Scatter was used for gating lymphocytes (A). The CD3+ population (B) was used to gate CD4+ and CD8+ cells (C). CD4+ T-cell subsets were identified based on CD45RA and CD27 expression (D). CD16+CD56+ NK cells were gated from CD3- cells (E). CD19+CD20+ B cells were gated from CD3- cells (F), and the HLA-DR expression of B cells was analyzed (G). CD14+ monocytes were identified based on scatter (H). CD3+ T cells, CD19+CD20+ B cells, CD16+CD56+ NK cells, and CD14+ monocytes were sorted by 4-way sorting. Post-sort analysis of each sorted population is shown (I–L).