Confocal microscopy.

Intravital microscopy was performed in vivo on calvaria and ex vivo on femurs with a confocal microscope (IV100 Olympus) as outlined in Online Figure I. A field of view at 4X magnification covered a 2290μm*2290μm, and 20X a 458μm*458μm dataset of 512 × 512 pixels. Z-stack images were acquired at 2μm steps. For in vivo imaging, mice were shaved at the skull and held in a stereotaxic skull holder. A skin incision revealed the calvaria, and glycerin (Sigma-Aldrich) was applied to prevent the tissue drying. The calvarian bone marrow was exposed as described before⁷. To delineate bone marrow cavities in the calvaria, OsteoSense® 750EX, a fluorescent bisphosphonate imaging agent, was administered intravenously 24 hours prior to imaging (4 nmol/mouse, NEV10053EX, PerkinElmer). IntegriSense™ 680, as an angiogenic marker for integrin αvβ3 activation, was administered intravenously 24 hours prior to imaging (2 nmol/mouse, NEV10645, PerkinElmer).

To outline the vasculature, we used FITC anti-CD31 (50μl, MEC13.3, 102514, Biolegend). To assess VEGFR2 activation, APC anti-VEGFR2 (Avas 12, 136406, Biolegend) was used in combination with IntegriSense 750™ (2 nmol/mouse, PerkinElmer, NEV10873). To analyze vascular leak, we followed a single plane through the center of the marrow cavities over 6min at a frame rate of 0.2083 frames/sec before and after bolus injection through an indwelling tail vein catheter of bovine serum albumin (BSA) labeled with rhodamine B and gadopentetic acid (GdDTPA) (RhoB-albumin-GdDTPA; excitation/emission: 555nm / 581nm, 82kDa, 2.5mg/mouse, Symochem, The Netherlands). For serial permeability imaging, FITC-albumin (495nm; 66kDa; 2.5mg/mouse; Sigma) was employed with the same temporal settings. To follow the cells serially by intravital microscopy of the calvarium, LSK (Lin-Sca-1⁺c-Kit⁺) were labeled ex vivo with the Vybrant® DiD Cell-Labeling Solution (1,1′-dioctadecyl-3,3,3′,3′- tetramethylindodicarbocyanine perchlorate, Molecular Probes®) according to manufacturer’s protocol. C57BL/6 mice were then intravenously injected with 75,000 labeled LSKs. In vivo imaging was performed using a confocal microscope (IV100 Olympus) on days 1 (day 0) and 2 (day 1) after adoptive cell transfer. Separately, 30min after intravenous injection of APC anti-Ly6G (1A8; Biolegend), Ly6G⁺ neutrophils were imaged on day 0 and day 1. For LSK and Ly6⁺ neutrophil serial imaging, LPS was injected intraperitoneally directly after the imaging session at day 0. After intravital microscopy, animals were sacrificed and femurs were dissected in 4% PFA and embedded in OCT. Femurs were shaved 300μm with a cryostat at −25°C to expose the bone marrow. These long bones were then thawed and imaged by confocal microscopy (IV100 Olympus) or by an epifluorescence imaging system (OV110 Olympus).