Hm48 1

Mitochondrial membrane potential within HSCs was quantified by flow cytometry as follows^{27 (link)}. Briefly, WBM cells from femurs and tibiae were depleted of lineage committed hematopoietic progenitors and surface stained for 30 minutes at 4⁰C with antibodies raised against SCA1 (D7; Biolegend), cKIT (2B8; Biolegend), CD150 (TC15-12F12.2; Biolegend), and CD48 (HM48-1; Biolegend). Following surface staining, cells were washed with MACS buffer and re-suspended in micro-centrifuge tubes containing 0.5 mL DMEM with TMRE (100 nM, ThermoFisher Scientific T669) and Verapamil (50 µM, Sigma-Aldrich V4629). Cells were incubated in a 37 °C CO₂ incubator for 25 minutes with caps open. Following incubation, cells were washed twice with ice-cold MACS buffer. Washed cells were re-suspended in ice-cold MACS buffer and maintained at 4 °C under low-light conditions until flow cytometry acquisition. Cells incubated in DMEM without TMRE served as gating controls. TMRE sensitivity was confirmed by collapse of TMRE intensity to background levels after mitochondrial uncoupling induced by 20 μM FCCP.

For HSC RNA isolation, lineage cell depletion was performed as described above and stained with antibodies raised against SCA1 (D7; Biolegend), cKIT (2B8; Biolegend), CD150 (TC15-12F12.2; Biolegend), and CD48 (HM48-1; Biolegend) and HSCs (defined as DAPI^-lineage^-SCA1⁺cKIT⁺CD150⁺CD48^-) were FACS sorted directly into TRIzol LS Reagent (ThermoFisher Scientific 10296010). For BMEC and LepR+ cell RNA isolation, mice were intravitally labeled with an antibody raised against VEcadherin (BV13; Biolegend) 10 minutes prior to sacrifice. Femurs and tibiae were gently crushed using a mortar and pestle and enzymatically dissociated with Digestion buffer for 15 minutes at 37 °C, filtered, washed in MACS buffer, and depleted of terminally differentiated hematopoietic cells as described under ‘BMEC and BM LepR+ cell isolation’. Depleted cell suspensions were stained with antibodies raised against CD45 (30-F11; Biolegend), TER119 (TER119; Biolegend), CD31 (390; Biolegend), and LEPR (R&D BAF497). Stained cells were washed in MACS buffer. BMECs (defined as CD45^-TER119^-Vecad⁺CD31⁺) and LepR+ cells (defined as CD45^-TER119^-VEcadherin^-LEPR⁺) were sorted directly into TRIzol LS Reagent. RNA was purified from TRIzol LS according to manufacturer’s recommendations.

Paired cells on the micropatterned antibody array were first imaged under FLIM mode using a Leica STELLARIS 8 FALCON inverted microscope with each cell’s location recorded. Cells were then washed with PBS and fixed with 4% paraformaldehyde (Electron Microscopy Sciences) in PBS at room temperature for 12 min. After washing with PBS, cells were blocked with 4% HyClone bovine serum albumin (BSA; Cytiva) in PBS at room temperature for 2 hours. Cells were then stained with Alexa Fluor 647–conjugated Tie2 antibody (clone 33, BD Biosciences; 1:100 dilution in 4% BSA) and phycoerythrin-conjugated CD48 antibody (HM48-1, BioLegend; 1:100 dilution in 4% BSA) at room temperature for 2 hours (26 (link)). Imaging of surface markers was carried out on the same microscope under the normal confocal mode.

WBM cells from femurs and tibiae were depleted of lineage committed hematopoietic progenitors and surface stained for 30 minutes at 4 °C with antibodies raised against SCA1 (D7; Biolegend), cKIT (2B8; Biolegend), CD150 (TC15-12F12.2; Biolegend), and CD48 (HM48-1; Biolegend). Following surface staining, cells were washed with MACS buffer and re-suspended in micro-centrifuge tubes containing 0.5 mL DMEM with CM-H2DCFDA (1 µM, ThermoFisher Scientific C6827). Cells were incubated in a 37 °C CO₂ incubator for 25 minutes with caps open. Following incubation, cells were washed twice with ice-cold MACS buffer. Washed cells were re-suspended in ice-cold MACS buffer and kept at 4 °C under low-light conditions until flow cytometry acquisition.