Supplementary Figure 13: Sorting strategy for in vitro differentiation of B cell and pDC progenitors. | Nature Methods

Supplementary Figure 13: Sorting strategy for in vitro differentiation of B cell and pDC progenitors.

From: FateID infers cell fate bias in multipotent progenitors from single-cell RNA-seq data

Supplementary Figure 13

(a) Sorting strategy for B cell versus plasmacytoid dendritic cell biased progenitors and staining of cultured progenitors. Cells were gated using SSC-A versus FSC-A (not shown). Only single cells were considered further on using SSC-W versus SSC-H gating followed by FSC-W versus FSC-H gating (not shown). Thereupon only lineage negative (Lin) cells were considered by exclusion of cells positive for the lineage markers TER-119, B220, Cd11b, Gr-1, SiglecH, Cd19 and Cd3ɛ (FITC). The next gating included only Flt3 positive (Flt3+) cells. The Flt3+ cells were plotted using Kit (BV510) versus Sca-1 (BV650) and cells with intermediate expression of Kit and Sca-1 were considered (common lymphoid progenitors, CLP). Finally CLP cells were plotted using IL-7R (BV421) and CD34 (PE). For the culture experiments and for single cell sequencing stringent gates were used to sort Cd34-IL7r+, Cd34+Il7r+ and Cd34+Il7r- cells. In addition, lymphoid-primed multipotent progenitors (LMPP) were sorted for culturing and for single cell sequencing. Fluorescence minus one controls were used for CD34 (PE) to set the sorting gates appropriately (not shown). The threshhold for Il7r-positive cells was set according to the unstained control (not shown). (b) Exemplary flow cytometry plots of progenitors from one mouse after 7 days of culturing in either B cell medium or plasmacytoid dendritic cell (pDC) medium. The surface marker expression of Siglech and Cd19 was assessed to check for lineage commitment towards the pDC or B cell lineage. Independent experiments were performed for n = 5 animals. Data for flow cytometry plot (a) is from one experiment with n = 3 mice, whereas data for flow cytometry plot (b) is derived from one experiment with n = 1 mouse.

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