Mouse cd31 microbeads

To isolate ECs, at experiment endpoint tumours were digested in cell lysis buffer (DMEM+2 mg ml⁻¹ collagenase type III) for 1 h at 37 °C. Up to 4 × 10⁷ cells were incubated with mouse CD31 microbeads (Miltenyi Biotec) and ECs were separated to a purity of ≥90% by positive selection according to the manufacturer's instruction. ECs (8 × 10⁵±1,3 × 10⁵) were isolated corresponding to 5.59%±1.45% of total cells.

Primary HUVECs were gained from ScienCell and cultured in ECM (ScienCell, 1001) supplemented with EC growth supplement (ECGS), 5% fetal bovine serum (FBS), and a penicillin/streptomycin cocktail. HUVECs were used for analysis before the 5th passage. mLECs were isolated as described. In brief, the lungs of adult mice were harvested and incubated with dispase (Roche, 10269638001). The homogenate was filtered through 100-μm and 40-μm cell strainers. The cell suspension was collected and incubated with mouse CD31 microbeads (Miltenyi Biotec, 130-097-418). The beads were washed with Dulbecco’s PBS (DPBS) supplemented with 1% FBS and then used for total RNA isolation. HEK293T cells were gained from the National Collection of Authenticated Cell Cultures and cultured in DMEM supplemented with 10% FBS. Wild type and TRPM7 KO T24 human urinary bladder carcinoma cells were generated in Dr. Zheng Zhang’s lab. The cells were authenticated through short tandem repeat (STR) by Genetic Testing Biotechnology Corporation (Suzhou, China) and cultured in DMEM supplemented with 10% FBS.

Skeletal muscle tissue (~500 mg) from the hind limb of 20-week-old mice was used. Tissue was manually cleaned of fat and cut into small pieces in DMEM. To combine enzymatic digestion and mechanical dissociation, a commercial murine skeletal muscle dissociation kit (Miltenyi Biotec, 130-098-305) was used in combination with the gentleMACS Octo Dissociator with heaters (Miltenyi Biotec, 130-096-427). Muscle pieces were transferred to C-tubes and digested with Miltenyi’s enzyme cocktail under agitation at 37°C for 61 minutes (program 37C_mr_SMDK_1). Samples were then filtered through a 70-μm strainer and washed with DMEM. To deplete CD45⁺ cells, the suspension was incubated for 15 minutes at 4°C under agitation with mouse CD45 microbeads (Miltenyi Biotec, 130-052-301) and then passed through an LS column (Miltenyi Biotec, 130-042-401). The cell suspension was then enriched for endothelial cells by incubating with mouse CD31 microbeads (Miltenyi Biotec, 130-097-418) for 15 minutes at 4°C under agitation and passed through an LS column. CD31⁺ cells retained in the LS column were eluted with PEB buffer (PBS, 2 mM EDTA, 0.5% BSA, pH 7.2) and centrifuged at 300g for 5 minutes. CD45^–CD31⁺ cells corresponding to endothelial cells were directly pelleted for RNA or protein isolation (71 (link)–73 (link)).

Four- to six-week-old C57BL/6 mice were anesthetized (ketamine/xylazine, 140/14 mg/kg), the thoracic cavity was exposed, and the lungs were removed. Isolated lungs were minced on a Petri dish and then incubated with 5 mL of collagenase A (2.5 mg/mL solution in 0.1% BSA in HEPES) in a 15 mL tube for 1 h in a 37 °C water bath with gentle shaking. The resulting tissue/cell suspension was filtered through a 70 μm strainer and washed twice with PBS. Cells were resuspended in an endothelial cell medium consisting of 4.5 g/L custom DMEM (PAN Biotech, Aidenbach, Germany, P04-03500S3), without L-glutamine and sodium pyruvate, with 3.7 g/L NaHCO₃ and D-valine, and without L-valine, supplemented with ECGS (Sigma-Aldrich, Saint Louis, MO, USA, E2759), 10% FBS, 2 mM L-glutamine, and 1% penicillin/streptomycin (v/v) in T-25 tissue culture flasks, and incubated in a CO₂ incubator. After reaching the specified density, the cells were sorted using mouse CD31 MicroBeads on a MACS column (Miltenyi Biotec, Bergisch Gladbach, Germany, 130-097-418), according to the manufacturer’s protocol. After sorting, cells were resuspended in an endothelial cell medium and passaged with the use of trypsin–EDTA. Cells below the fifth passage were used for experiments.

Primary PMVECs were isolated using CD31 MicroBeads mouse according to the manufacture’s protocols provided by Miltenyi Biotec (Bergisch Gladbach, Germany). Briefly, lungs from wild type and RAGE knockout mice were excised, sliced, digested in collagenase type I, and then filtered through 70 μm nylon filters. Centrifuge cell suspension at 300 × g for 10 minutes. Move out supernatant and add 10 μl of CD31 microbeads per 10⁷ total cells. Mix well and incubate for 15 min in the refrigerator (2 °C–8 °C). Place column in the magnetic field of a suitable MACS Separator. Apply cell suspension onto the column and collect unlabeled cells that pass through the column and repeat I for several times. Finally, remove column from the separator and place it on a suitable collection tube, flushing out the magnetically labeled cells into it. Purity of mouse PMVECs were evaluated by factor VIII labeling.

The isolation of BMECs was performed as previously described with minor revision. Summarily, the bone marrow was collected from the tibiae and femurs of mice using a sterile liver digestion medium (Gibco) supplemented with 0.1% DNaseI (Invitrogen). To obtain single BM cell suspension, BM was digested for 30 min at 37 °C under shaking conditions. BMECs were then sorted using CD31 MicroBeads, mouse (Miltenyi Biotec). Sorted BMECs were seeded on fibronectin (Sigma–Aldrich) coated dishes and cultured in endothelial cell medium (ECM, Sciencell) supplemented with endothelial cell growth supplement, fetal bovine serum, and antibiotic solution (Sciencell) at 37 °C 5% CO2. At first passage, BMECs were sorted using CD31 MicroBeads and plated for culture. The culture medium of BMECs was changed every other day and passaged upon confluency. Passages 2 to 4 of BMECs were used for subsequent experiments.