Anti mouse igk and negative beads

Cells were stained with PI/DAPI (BD Biosciences, RUO) and antibodies at 1:100 dilution in PBS with 2% FBS for 30 min at room temperature in the dark. BD LSRII and BD FACSAria II were used for flow cytometry analysis and cells sorting. Compensation was performed by automated compensation with anti-mouse Igk and negative beads (BD Biosciences) using the BD FACSDiva software. The following human antibodies were used: CD45RA-BV510 (BD Biosciences, H100), CD45RO-PE (BD Biosciences, UCHL1), CCR7-APC (BD Biosciences, 2-L1-A), CD3-PE/Cy7 (Biolegend, SK7), CD8α-BV421 (BD Biosciences, RPA-T8), TCRαβ-APC (Biolegend, IP26), TCRγδ-PE (Biolegend, B1), CD7-PE (BD Biosciences, M-T701), CD5-BV510 (BD Biosciences, UCHT2), CD4-PE/Cy5 (Biolegend, RPA-T4), CD8β-APC (Miltenyi, REA715), CD45-APC/Cy7 (BD Biosciences, 2D1), CD279-BV421 (BD Biosciences, EH12.1), CD366-APC (BD Biosciences, F38-2E2), CD223-PE (BD Biosciences, T47-530), CD107a-PE (Biolegend, LAMP-1), CD69 APC (Biolegend, FN50), CD33-APC (Biolegend, WM53).

Human erythropoiesis, including differentiation from BM CD34+ cells and native bone marrow samples, was analyzed with the following antibody panel: CD71 PE (M-A712; BD), and CD235a/Glycophorin A PE- Cy7 (11E4B-7–6; Coulter) or CD235a/Glycophorin A FITC (11E4B-7–6; Coulter). Mouse erythropoiesis from Neratinib treated and HER4^heart mice was analyzed with the following antibody panel: mCD71 FITC (C2; BD), mTer119 PE-Cy5 (Ter-119; eBioscience). All staining was performed with < 1×10⁶ cells per 100 μL staining buffer (PBS + 2% FBS) with 1:100 dilution of each antibody for 30 min at RT in dark. Compensation was performed by automated compensation with anti-mouse Igk and negative beads (BD). Acquisition was performed on a BD Fortessa cytometer and all sorting was performed on a BD FACS Aria II cell sorter using a 70-mm nozzle. Gating strategies are depicted in Supplementary Fig. 15.

Mice were sacrificed after 14 weeks, and injected femur, and uninjected femur and tibiae were collected. Single cell suspension was prepared using standard flushing and cell dissociation techniques in 1 ml of IMDM. From that suspension, 100 μl of injected femur, 50 μl uninjected marrow (~1×10⁶ cells) were stained in a total volume of 200 μl staining buffer. Samples were not lysed with red blood cell lysis buffer as not to lyse human erythrocytes. Samples were stained with the 1:75 dilution the following antibodies: SytoxGreen live-dead stain (Thermo), CD33-PE (WM53; BD), CD45-APC (P67.6; BD), CD235a (11E4B-7–6; Coulter), CD19-BV605 (SJ25C1; BD), CD3-BV786 (SK7; BD), and CD66b-BV421 (G10F5; BD). All antibodies were confirmed as human-specific. Uninjected mouse marrow was used as a control for non-specific staining; CB mononuclear cells were used as a positive control. Compensation was performed using automated compensation with anti-mouse Ig^k and negative beads (BD Biosciences).