Mck00

SCF and CXCL12 from serum and BM fluid were measured using ELISA kits (MCK00 from R&D Systems and MCX120 from R&D Systems, respectively). Briefly, to collect BM fluid, a small hole was made at the tip of the long bones before spinning the BM out of the bones by centrifugation at 6000g for 6 minutes. Supernatant was then collected. IL-3 and TPO from serum was measured using ELISA kits (EK0403 from BosterBio and MTP00 from R&D Systems). IL-6 and TNF-α from plasma were measured using ELISA kits (M6000B from R&D Systems and MTA00B from R&D Systems, respectively). To quantify soluble NETs (citrullinated histone H3–DNA complexes), a custom ELISA was used. For antibody capture, an anti-citrullinated histone H3 antibody (ab5103, Abcam) was used. Detection of complexes was done using anti–DNA-HRP conjugate (Cell Death Detection ELISA^plus Kit, Roche; ref. 45 (link)).

Plasma was collected from whole blood centrifuged at 2000g for 20 min. cBM fluid was collected by flushing leg bones in 250 µl PBS. Flushed bones were then crushed in 200 µl PBS to collect eBM fluid. Lysate samples were generated by lysing sorted cells or crushing in lysis buffer (1% NP40, 1× Protease inhibitors (Roche, Switzerland), 1 mM AEBSF, 1 mM Na₃VO₄, 50 mM TrisHCl, 150 mM NaCl, 1 mM EDTA). MK conditioned media was generated from the culture of 2000 LCM or SCM in 100 µl IMDM, 1% BSA (Sigma) and 5 ng/ml hTPO for 3 days. ELISAs were performed as per the manufacturer’s instructions (PF4 (MCX400, R&D Systems, USA), TPO (MTP00, R&D Systems, USA), FX (MFXKT-TOT, Molecular Innovations, USA), TGFβ1 (MB100B, R&D Systems, USA), SCF (MCK00, R&D Systems, USA), MMP-9 (MMPT90, R&D Systems, USA), SDF-1 (MCX120), FGF-1 (Abcam, ab223587, USA) and tcOPN (27259, IBL, Japan) and data were recorded using an EnSpire 2300 Multilabel Reader, EnSpire Manager 4.13.3005.1482 (PerkinElmer) was used to assess ELISA results.