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. 2014 Feb 14;9(2):e88311. doi: 10.1371/journal.pone.0088311

Figure 6. LT-HSC in neonatal spleen contribute to DC production in stromal co-cultures.

Figure 6

(A) Spleen cells isolated from one-day old C5BL/6J mice were stained with antibodies to distinguish hematopoietic progenitors flow cytometrically. A lineage cocktail of antibodies (CD3, CD4, CD5, CD8, B220, Gr-1, CD11b, Ter119, CD11c) was used to exclude mature Lin+ cells. Staining with Propidium iodide (PI:1ugm/ml) was used to delineate PI− live cells. Sca-1 and c-kit staining was used to identify the Lin−c-kit+Sca-1+(LSK) subset. Staining with Flt3 and CD150 was used to distinguish and sort LT-HSC and MPP, which were overlaid in co-cultures above 5G3 stroma. (B) Non-adherent cells produced in co-cultures were stained with antibodies to detect a CD11b+CD11c+ population of dendritic-like cells. These were tested for expression of MHC-II, 4-1BBL, F4/80, CD24, CD8, and B220 to delineate L-DC and cDC-like subsets. Data show cell production at 21 days for one of three replicate cultures established from different mice. Gates were set on bivariate plots using isotype control antibodies and numbers on gates reflect % positive cells. (C) Production of CD11b−CD11c− progenitors and CD11b+CD11c+ dendritic-like cells was calculated in terms of proportion of each subset and number of cells of each type produced. Graphs show mean±S.E. for triplicate co-cultures.