48 well plate

Before experiments, 48‐well plates (Millipore), matrigel (Corning), and pipette tips were precooled or dissolved at 4°C. matrigel (100 μl) was pipetted into a 48‐well plate with a precooled tip, and the plate was put in an incubator (37°C, 30 min). HUVECs (2 × 10⁴) with different treatments were seeded into each well. After 6 h of cell culture, photographs were taken and vessel length was analyzed by ImageJ software (NIH).

In order to release ALP from cells, the following steps were performed according to the protocols developed by Thanih et al. [42 (link)]. The first step was to enhance ALP release in 4-day-old culture by seeding cells as a monolayer on 48-well plates (Sigma, Ireland), incubating at 37 °C and 5% CO₂ (incusafe Panasonic incubator) and changing media every two days. The cell lines used during this study were purchased from ATCC (UK), including mouse embryo fibroblast cells (Balb/c 3T3 Line), breast carcinoma epithelial cells (MCF-7 Line), lung carcinoma epithelial cells (A-549 Line), and colon carcinoma epithelial cells (Ht-29 Line). The media, supplements, and washing buffer used for culturing cells were Dulbecco’s modified Eagle’s medium (DMEM), minimum essential medium Eagle (MEME) and McCoy’s 5A medium, newborn calf serum (NBCS), fetal bovine serum (FBS), and Hank’s balanced salt solution (HBSS), which were purchased from Sigma (Ireland). Table 1 summarises the cell numbers used and the composition of media. The cells were sub-cultured three times before seeding began under aseptic conditions using a cell culture hood (Esco Airstream^® Class II).

L-aspartic acid (Sigma-Aldrich, UK), 1,4-diaminobutane (DAB) (Sigma-Aldrich, ≥99%), cysteamine (CYSE) (Sigma-Aldrich, UK), cystamine (CYS) (Sigma-Aldrich, UK), dimethylformamide (DMF) (VWR International, USA), dimethylsulfoxide (DMSO) (Sigma-Aldrich), o-phosphoric acid (VWR), imidazole (ACS reagent, ≥99%, Sigma-Aldrich), citric-acid*H₂O (ACS reagent, ≥99.9%, VWR), sodium chloride (99–100.5%, Sigma-Aldrich), phosphate buffer saline (PBS) (Tablet, Sigma), D,L-dithiotreitol (DTT) (Sigma), 5,5 dithio bis-(2-nitrobenzoic acid) (Sigma, ≥98%, USA), L-cystein (Sigma, ≥97%, USA), Humidified incubator (Nuaire, USA), 100 mm tissue culture dishes (Orange Scientific, Belgium), 48 well plates (Sigma-Aldrich, USA), low cell binding 96 well plates (Nunc, Denmark), Eagle’s Medium Alfa minimal essential medium (αMEM) (Gibco, USA), fetal bovine serum (FBS, Gibco, USA), L-glutamine (Gibco, USA), penicillin and streptomycin (Gibco, USA), L-ascorbic acid 2-phosphate (Sigma-Aldrich, USA), beta-glycerophosphate (Sigma-Aldrich, USA), dexamethasone (Sigma-Aldrich, USA), WST-1 [2-(4-Iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium] (Roche, Switzerland), Vybrant DiD (Molecular Probes, USA), 2-Amino-2-Methyl-1-Propanol buffer (Sigma-Aldrich, USA), Alkaline Phosphatase Yellow (pNPP) Liquid Substrate System (Sigma-Aldrich, USA).

Individual neurospheres were collected and plated into laminin-coated wells in a 48 well plate (Sigma-Aldrich). Each sphere was plated in NBM containing 1% fetal bovine serum (FBS; Wisent Bioproducts, QC, Canada) and 100 ng/mL metformin for 7 days at 37 °C and 5% CO_2, then fixed using ice-cold 4% paraformaldehyde (PFA) for 20 min, rinsed in PBS and stored at 4 °C until processed for immunohistochemistry.