Pore transwells

For the transendothelial migration assay,VeraVec^TM mouse spleen ECs (Angiocrine Biosciences) were cultured to confluence in 8 µM pore transwells (Corning Incorporated). Transwells were then seeded with 50,000 sorted BM KSL cells in IMDM with 10% FBS, 1% pen-strep, 20 ng/mL TPO, 125 ng/mL SCF, and 50 ng/mL Flt3 ligand with or without 1 µg/mL of DJ001. SDF-1, 500 ng/mL (R&D Systems), was added to the bottom chamber of the transwell. At 18 h post incubation, cells in the bottom chamber were collected and cell counts were performed.

Transwell assays were performed according to the manufacturer’s protocol to determine whether MSCs could enhance the migration of BMDMs. Each MSC line was suspended in DMEM/FBS/PS on a 24-well plate (1.5 × 10⁵ cells/500 µL) and incubated for 4 h until the cells adhered to the bottom surface. Pore transwells (8.0 µm, Corning, USA) were set in the wells and seeded in upper layers with BMDM (1.5 × 10⁵ cells). Following incubation for 24 h, the number of migrating cells was counted. The transwells were collected and fixed with 4% PFA for 20 min and washed with PBS, and the upper surface of the membrane was cleared. The membrane was cut and attached to a glass slide and mounted with Vectashield containing DAPI (Vector Laboratories, USA). Images of the entire membrane were captured with a BZ-9000 fluorescence microscope (Keyence, Japan), and the images were merged with a BZ-II Analyzer (Keyence). The combined images were used to measure the number of cells using the ImageJ software [16 (link)].

HBE progenitors were isolated from donor airways as described previously, grown for a week to confluency and frozen for later use as described [22 (link)]. Progenitor cells were thawed and plated on 0.4 μM pore Transwells (Corning) membranes, 6.5 mm or 12 mm in diameter, fed with ALI medium supplemented with ROCK inhibitor in both the apical and basolateral chambers. Medium in both chambers was replaced with fresh medium every 2–3 days. At 7 days, when the cells were confluent and had formed tight junctions as demonstrated by electrical resistance, the apical medium was removed, and the basal medium was replaced with complete Pneumacult-ALI Medium (STEMCELL Technologies). The medium was replaced with fresh medium and the apical surface was washed with 100 μL of DMEM every 2–3 days for 3 weeks by which time they had become fully differentiated.