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. 2019 May 22;14(5):e0217163. doi: 10.1371/journal.pone.0217163

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© 2019 Jimenez Vera et al

This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

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Fig 3 — (A) For panel 2, the first gate (G1) is on time vs CD45 to exclude poor flow, the second gate (G2) is on CD45 vs SSC-A to exclude granulocytes, the third gate (G3) is on FSC-A vs SSC-A for separating monocytes, including DCs (G3a), and lymphocytes (G3b). Under G3a is G4a which is on FCS-A vs FSC-H to exclude double-cells, G5a is on CD16 vs CD45 to exclude granulocytes again, G6a is on CD3 vs CD19 to exclude T and B cells, G7a is on CD14 vs SSC-A for CD14+monocytes, with further assessment of classical (CD14+brightCD16-), intermediate (CD14+brightCD16+), non-classical (CD14+dimCD16+bright); CD16+CD64+(CD16 vs CD64); and CD56+CD16- (CD16 vs CD56) monocytes. Under G3b is G4b which is on FCS-A vs FSC-H to exclude double-cells on lymphocyte. CD3 vs CD19 under G4b is for B and T cells populations. Gating on CD3+ T cells (G5b) was done to further assess CD4+, CD8+, CD4-CD8- and CD4+CD8+ subsets (CD4 vs CD8); gating on CD19- cells (G6b) was undertaken to assess CD3+CD56+ NKT cells and CD56+ NK cells (CD56 vs CD3); gating on CD3+CD56+ NKT (G7b) was done for assessment of CD4 vs CD8 subsets of NKT cells. Gating on CD56+ NK in gate G8b further assessment of CD16+CD56+, CD16+CD56+bright (intermediate), CD16-CD56+bright and CD16-CD56+ NK subsets (CD56 vs CD16), and CD4+, CD8+, and CD4-CD8- NK subsets (CD4 vs CD8) was undertaken. (B) The gating strategies for the DC panel (panel 3). The first and second gates were the same as in panel 2. Gating on HLA-DR+LIN- (CD3CD14CD19CD20CD56) in gate G3 (LIN vs HLA-DR) was performed to exclude CD14+ monocytes, CD3+ T, CD19+ and CD20+ B, CD56+ NK cells, then the fourth gate (G4) was performed to exclude double-cells. Gating on CD11c+HLA-DR+ cells in gate G5 was performed to assess CD141+mid/bright mDC2, CD141- mDC1 and CD16+ DCs (pseudocolor and smooth of CD141 vs CD16); and gating on CD11c-HLA-DR+ cells (G6) was undertaken to assess plasmacytoid DCs CD303+CD123+ (pDCs) and CD303-CD123-/dim HLA-DR+ immature cells (CD303 vs CD123).