Cd31 magnetic beads

For isolating ASCs from endothelial cells, CD31 magnetic beads (Miltenyi Biotech, Bergish, Gladbach, Germany) and a miniMACs magnet kit (Miltenyl Biotech) were used [8 (link)]. Primary ASCs cultures (1 × 10⁷ cells) were trypsinized and centrifuged at 300×g for 5 min. Cells were then recovered in 60 μL of GM. After blocking from 20 μL of FcR blocking reagent, 20 μL of CD31 microbeads were added to the cells and incubated at 4 °C for 15 min. The microbeads were applied to the magnetic column. The unlabeled cells (CD31⁻) were collected, whereas the labeled cells (CD31⁺) were discarded. In this study, CD31⁻ ASCs were called ASCs, and subsequently seeded at 1 × 10⁴ cells per 60-mm² dish (Falcon).

The method of tissue digestion was the same as that of the single-cell sample preparation, the difference was that the digestion time for ECs was 20 min, while that of FB cells was about 40 min. The digested single-cell suspensions were washed and re-suspended in 10% FBS/DMED (Gibco) and EGM-2 (CC-3202; Lonza) medium for FB and EC culturing, respectively. Because of the strong proliferation capacity of FBs, FB cells would eventually dominate in DMEM medium, and the purity was more than 95% after passage. For ECs, cell cloning began at about day 5 of culture, then fibroblast inhibitors were added for 7–10 days. When isolated colonies began to fuse with each other, the EC colonies were marked and digested using a clonal cylinder (C7983-50EA; Sigma-Aldrich), and the EC suspension was further purified using CD31 magnetic beads (130-091-935; Miltenyi).

Fresh CC tissue samples of approximately 1 × 1 cm size were first immersed in 15 mL PBS with vigorous shaking to remove residual blood cells. Then, tissues were cut into pieces of 1 × 1 mm size and enzymatically digested with 2.5 mg/mL collagenase type I (17104-019, gibco, US), 4 mg/mL collagenase type IV (17018029, gibco), 0.1 mg/mL neutral protease, and 2 mg/mL DNase I (#AMPD1, Sigma-Aldrich, US) at 37 °C for 20 min. Subsequently, the cell suspension was filtered through a 40 mm nylon mesh, and the cells were sorted by MACS with a Dead Cell Removal Kit (130-090-101, Miltenyi Biotec, Germany) to remove dead cells. Briefly, 10⁶ cells were co-incubated with 20 µL MicroBeads at 4 °C for 30 min. After blocking and washing, magnetic separation with LS Columns (130-042-401; Miltenyi) and MidiMACS Separator (130-042-302; Miltenyi) was performed, retaining cells that were not adsorbed by the column. CCECs were cultured with EGM-2 medium and cell cloning began at approximately day 5 of culture, then fibroblast inhibitors were added for 7–10 days. When isolated colonies began to fuse with each other, the EC colonies were marked and digested using a clonal cylinder (C7983-50EA; Sigma-Aldrich), and the EC suspension could be further purified using CD31 magnetic beads (according to their purity) (130-091-935; Miltenyi).

Primary mouse LN LECs were isolated as previously described [28 (link)]. Briefly, popliteal, inguinal, axillary, brachial, and auricular LNs were isolated from 8–12-week-old animals; torn; and digested in RPMI medium (Thermo Fisher Scientific) containing 0.25 mg/mL Liberase TL (Roche, Basel, Switzerland) and 1 mg/mL DNase I (Roche) at 37 °C for 1 h. After 15 min, the LNs were cut with scissors, and thereafter further disrupted by pipetting every 10–15 min. The cell suspension was passed through a 70-µm cell strainer, centrifuged, and resuspended in full LN LEC medium (αMEM (no nucleosides, Thermo Fisher Scientific), 10% FBS, 1% penicillin-streptomycin (Thermo Fisher Scientific), and 1% L-glutamine (Thermo Fisher Scientific)). Cells were cultured at 37 °C and 5% CO₂ in dishes precoated with 10 µg/mL collagen (PureCol, Advanced BioMatrix, Carlsbad, CA, USA) and 10 µg/mL human fibronectin (Merck, Darmstadt, Germany) in PBS. Nonadherent cells were washed away with warm PBS on day 1 and day 3. Once the cells reached about 80% confluency, they were detached with accutase (Sigma-Aldrich) and purified with CD31 magnetic beads (Miltenyi Biotec, Bergisch Gladbach, Germany) according to the manufacturer’s instructions. Purified LECs were cultured in full medium on coated plates and used for experiments in passages 3–5.