70 μm falcon cell strainer

Cord blood (CB) units were purchased from King Abdullah International Medical Research Center’s (KAIMRC) Cord Blood Bank. CB buffy coat-enriched cells were thawed in MEM-ɑ medium containing 10% ACD-A (anticoagulant citrate dextrose solution) and 5% human serum albumin (Sigma), centrifugated at 300g, resuspended in 10 mL PBS containing 0.1 mg/mL DNAse, incubated at room temperature for 10 minutes, centrifuged, washed twice with MEM medium containing 10% ACD-A, resuspended in 30 mL of MEM-ɑ culture medium with 5% FBS and incubated at 37°C for 12 to 24 hours in a hypoxic chamber (Stem cells Technologies) containing water in a petri dish. The following day, the cells were rinsed with PBS/ACD-A, exposed to a density gradient using Ficol-paque (GE Healthcare) in LeucoSep™ centrifuge tubes (Thomas Scientific) for 12 minutes at room temperature with brake-off, then the mononuclear layer was transferred to a separate tube, washed twice with PBS/ACD, filtered using a BD Falcon 70 μm cell strainer to obtain single cells, which were enumerated, stained, sorted, and subjected to flow cytometric analysis.

For endothelial cell isolation the resected specimens containing normal or malignant tissue were dissociated using a standardised, semi-automated protocol based on a combination of mechanical tissue disruption and collagenase IV digestion, using a gentleMACS dissociator (Miltenyi Biotec, Bergisch Gladbach, Germany). Briefly, tissues were cut in small pieces (2–4 mm) in a petri dish prior to collection in a dedicated gentleMACS tube, to which 10 ml of RPMI 1640 (Lonza, BioWhittaker, Walkersville, MD, USA) with 1% L-Glutamin, 1% Penicillin/Streptomycin, 10% heat-inactivated FBS (Sigma Aldrich, St Louis, MO, USA), as well as collagenase IV (Sigma Aldrich), 1 mg ml⁻¹, and DNAse I (Sigma Aldrich), 10 μg ml⁻¹, had been added. The tissues were mechanically minced on the gentleMACS after incubation for 30 min at 37 °C in a shaking water-bath. This procedure was repeated once. After the second dissociation step at the gentleMACS, the cell suspension was passed through a 70 μm Falcon cell strainer (BD Biosciences, San Jose, CA, USA). The cell suspension was then collected in a 50 ml conical tube, and centrifuged at 1000 g for 5 min. The resulting pellet was washed with 50 ml phosphate buffered saline (PBS) and concentrated at 2 × 10⁶ cells ml⁻¹ in PBS for flow cytometry to detect and characterise the tissue-derived ECs.

On day 21 post tumor inoculation, tumor-bearing mice were sacrificed, tumors were isolated, weighed, minced into small pieces and re-suspended in a 37°C pre-warmed digestion medium composed of 1 mg/mL Collagenase A (Roche) in WiIliam's E medium (Gibco). The gentleMACS™ Dissociator (Miltenyi Biotec) was used to homogenize tumors, according to the manufacturer's protocol. Homogenized tumors were then incubated for 30 min at 37°C on a shaker (120 rpm). The homogenization and incubation procedures were repeated once more and then cells were filtered through a 70 μm Falcon cell strainer (BD Bioscience). Sterile lysis buffer [(150 mM NH₄Cl, 10 mM KHCO₃, 0.1 mM Na₂-EDTA pH = 7.2–7.4), in a volume of 5 mL per sample] was used to lyse erythrocytes for 5 min at room temperature. 15 mL Iscove's modified Dulbecco's medium (IMDM) per sample was used to stop the lysis reaction. Cell suspensions were then centrifuged, and the supernatant was discarded and cells resuspended in IMDM with 5% FCS.