Isolating Bone Marrow Dendritic Cell Progenitors

Bone marrow progenitors and DCs were isolated as described^{9 (link)}. For BM sorting experiments, BM was isolated and depleted of CD3-, CD19-, CD105-, Ter119-, and in some instances Ly6G- and CD45R-expressing cells by staining with the corresponding biotinylated antibodies followed by depletion with MagniSort Streptavidin Negative Selection Beads (Thermo Fisher). All remaining BM cells were then stained with fluorescent antibodies prior to sorting. MDPs were identified as Lin⁻CD117^hiCD135⁺CD115⁺ BM cells; CDPs were Lin⁻CD117^intCD135⁺CD115⁺MHC-II⁻CD11c⁺; pre-cDC1s are Lin⁻CD117^intCD135⁺CD115⁻MHC-II^lo-intCD11c⁺CD24⁺Siglec-H⁻ or as Lin⁻CD117^intCD135⁺MHC-II^lo-intCD11c⁺Siglec-H⁻Zbtb46-GFP⁺, and pre-cDC2s as Lin⁻CD117^loCD135⁺CD115⁺MHC-II⁻CD11c⁺. For splenic sorting experiments, spleen was isolated and depleted of Ly6G-, B220-, and CD3-expressing cells. cDC2 were identified as Lin⁻CD45R⁻CD317⁻MHC-II⁺CD11c⁺CD172a⁺ cells. Cells were purified on a FACSAria Fusion into IMDM plus 10% FBS with 5% Flt3L conditioned media. Sort purity of >95% was confirmed by post-sort analysis before cells were used for further experiments. For experiments that included Flt3L cultures, sorted cells (1×10³ to 10×10³ cells per 200 µl complete IMDM) were cultured for 5 or 7 d at 37 °C with 5% Flt3L conditioned media.