12865_2018_279_MOESM6_ESM.tif (8.74 MB)
Download file

Additional file 6: of Human-like NSG mouse glycoproteins sialylation pattern changes the phenotype of human lymphocytes and sensitivity to HIV-1 infection

Download (8.74 MB)
posted on 2019-01-07, 05:00 authored by Raghubendra Dagur, Amanda Branch-Woods, Saumi Mathews, Poonam Joshi, Rolen Quadros, Donald Harms, Yan Cheng, Shana Miles, Samuel Pirruccello, Channabasavaiah Gurumurthy, Santhi Gorantla, Larisa Poluektova
Figure S6. Humanized bone marrow gating strategies. The percentage of lymphocytes was enumerated based on two CD45 by light scatter displays so that cells had to be present in both gates to be considered lymphocytes. T-cells, NK-cells, and monocytes were enumerated using a T/NK-cell cocktail containing CD3, CD4, CD7, CD8, CD14, CD16, and CD56. T-cells were identified as CD3+, low side light scatter events and were further characterized for CD4 and CD8 expression. NK-cells were isolated using a low side light scatter (SS) gate on the CD45 by side light scatter histogram. The low SS cells were characterized for CD56 and CD16 expression to enumerate the two NK-cell subsets (not shown here). Monocytes were isolated using a Boolean logic gate as CD4dim and CD3neg cells. Promonocytes and mature monocytes were identified based on CD14 expression density. CD19+, CD10+, and CD20+ B-cells were enumerated using a B-cell specific cocktail containing kappa, lambda, CD10, CD19, CD20, CD24, and CD38. CD19-positive, low SS cells were gated to enumerate total B-cells and precursors. The CD19-positive B-cells were characterized for expression of CD10 to identify the B-cell precursors and CD20 to identify transitional to mature B-cells. Myeloblasts, proB-cells, preB-cells, mast cells, and granulocytes were enumerated using a myeloid cell cocktail containing HLA-DR, CD10, CD13, CD24, CD33, CD34, and CD117. CD34-positive events were gated on a CD34 by SS histogram and were characterized as myeloblasts or pro B-cells based on the CD45 by SS profile. Total B-cell precursors were isolated based on HLA-DR, CD10, and CD24 co-expression. PreB-cells were calculated using Boolean logic as total B-cell precursors and not proB-cells. Mast cells were enumerated as CD117bright events on a CD34-positive by CD117 display. Finally, the granulocytes were estimated based on a CD45 by high SS gate that excluded the CD117bright mast cells. (PDF 1680 kb)


NIH Office of the Director