Myjector syringe

For single-cell transplantations, single Vwf⁺ or 5 Vwf⁻ HSCs (CD45.2 allotype) were FACS-sorted using the FACSAriaII automated cell deposition unit, and collected into individual wells of a round-bottomed 96-well plate containing 100 μl of transplantation media: Iscove's modified Dulbecco's medium (IMDM) containing 1% penicillin–streptomycin, 1% β-mercaptoethanol, 1% L-glutamine and 20% BIT 9,500 serum substitute (Stem Cell Technologies). To ensure that single cells were deposited, single fluorescent beads were sorted, and after microscopic inspection >98% of the wells were found to contain a single bead, and no wells more than one bead. After sorting single cells, 1 million W⁴¹/W⁴¹ (c-Kit-deficient; CD45.1 allotype) unfractionated competitor BM cells in 100 μl volume were added to each well. Cells were mixed, loaded into a Terumo Myjector syringe and injected into the lateral vein of lethally irradiated (1,050 cGy, split dose, 4 h apart) recipients (CD45.1 allotype). Peripheral blood cells from recipient mice were analysed at 6, 10 and 16 weeks post-transplantations.

The TJ barrier function was assessed in vivo using a previously described biotinylation technique²⁶, ²⁷, ²⁸ with some modifications. First, 50 μL of 10 mg/mL EZ‐Link Sulfo‐NHS‐LC‐Biotin (21355; Thermo Fisher Scientific) in PBS containing 1‐mM CaCl₂ (C‐34006; PromoCell, Heidelberg, Germany) was injected by Myjector syringe (SS‐05M2913; Terumo Corporation, Tokyo, Japan) into the dorsal ear of the C57BL/6J mouse under anaesthesia. After 60 min, we sacrificed the mouse and collected ear samples. In the 3D model, the insert was turned upside down and the tracer solution was applied to the bottom side. Sixty‐minutes later, cell sheets were collected. We soaked in vitro and in vivo samples in a frozen section embedding agent (Cryomount I, 33351; Muto Pure Chemicals) and sectioned them with a cryostat (Leica CM1950, Leica Biosystems). The staining procedure was performed as described above, except that streptavidin Texas Red (dilution 1:200 in PBS 189738; Merck, Darmstadt, Germany) was additionally mixed into the solution of secondary antibodies. Fluorescence microscopic images were taken (BZ9000, BioRevo; Keyence).