HSCs and LMPPs were isolated as previously described (44 (link)). Briefly, lineage depletion was carried out by removal with magnetic beads conjugated to BioMag goat anti-rat IgG (Qiagen 310107) of the following marker antibodies: Ter119, B220, CD19, Mac-1, Gr-1, IgM, CD3, CD8a, and DX5. Lineage-negative cells were stained with the following antibodies: HSC (Lin−, cKit+, Sca-1int/+, CD34+, FLT3−) and LMPP (Lin−, cKit+, Sca-1int/+, CD34+, FLT3+).
ALPs (or CLPs) and BLPs were isolated as previously described (46 (link)–48 (link, link)). Briefly, after lineage depletion (Ter119, Mac-1, B220, Gr-1, CD3e; biotin anti-mouse lineage panel Biolegend 133307), bone marrow cells were stained as followed: ALP (Lin−, cKitmid/lo, IL7Ra+, Sca-1lo, FLT3+, Ly6D−) and BLP (Lin−, cKitmid/lo, IL7Ra+, Sca-1lo, FLT3+/int, Ly6D+).
Pro-B, large pre-B, and small pre-B cells were isolated as previously described (49 (link), 50 (link)). Bone marrow cells were stained as followed: pro-B (CD19+, IgM−, cKitlo, CD43+), large pre-B (CD19+, IgM−, cKit−, CD43+/−, CD2−), and small pre-B (CD19+, IgM−, cKit−, CD43−, CD2+).
Lung epithelium cells were stained as followed: CD326 (EpCam)+, CD19−, CD3−, and CD45−.
Postsort analysis confirmed purity of cell fractions.
ALPs (or CLPs) and BLPs were isolated as previously described (46 (link)–
Pro-B, large pre-B, and small pre-B cells were isolated as previously described (49 (link), 50 (link)). Bone marrow cells were stained as followed: pro-B (CD19+, IgM−, cKitlo, CD43+), large pre-B (CD19+, IgM−, cKit−, CD43+/−, CD2−), and small pre-B (CD19+, IgM−, cKit−, CD43−, CD2+).
Lung epithelium cells were stained as followed: CD326 (EpCam)+, CD19−, CD3−, and CD45−.
Postsort analysis confirmed purity of cell fractions.
