Matrigel basement membrane matrix growth factor reduced

NSCLC CSCs were clonally expanded for 1 week. Spheres were gently suspended in 20% Matrigel and were seeded into 96-well plates onto a layer of 35 μL of Matrigel Growth Factor Reduced Basement Membrane Matrix (BD Biosciences). Once solidified, CSC culture medium was placed on top of the cells. Spheres were visualized and images were captured microscopically (Olympus microscope) 5 days after plating and photographed to observe cellular morphology.

A stock solution of bovine fibrinogen (Sigma: F8630) was prepared in sterile PBS (100 mg/ml) and stored at -20°C. The stock solution was diluted to 5 mg/ml in proliferation medium. Fibrinogen was then mixed with cells or muscle fibres and Thrombin (Tissuco Duo 500, Baxter, Illinois, USA) was added on ice at a final concentration of 3 U/ml. The solution was quickly mixed and incubated at 37°C for 30–60 mins to polymerise. To avoid Fibrin degradation, Aprotinin (Sigma A1153) was added at a final concentration of 33 μg/ml in culture medium (Table 1). Polymerised Fibrin gel formed using 5 mg/ml of Fibrinogen and 3U/ml of Thrombin has a stiffness of ~1±0.1 KPa [67 (link)] [68 (link)]. As a positive control for some experiments, 10% (volume) of Matrigel (Growth Factor Reduced Basement Membrane Matrix) (BD 354230) was added to the Fibrin gel to a final concentration of ~1 mg/ml.

The tube formation ability of HBECs was assessed using Matrigel^® Growth Factor Reduced Basement Membrane Matrix (BD Biosciences). For this purpose, 2 × 10⁴ cells/well were seeded onto the plated Matrigel separately in six different media: endothelial basal media (EBM) (ATCC), conditioned media from U87_TRAF3IP2KD or U118_TRAF3IP2KD cultures (U87_TRAF3IP2KD CM or U118_TRAF3IP2KD CM, respectively), and conditioned media from U87_{control shRNA} or U118_{control shRNA} cultures (U87_{control shRNA} CM or U118_{control shRNA} CM, respectively) in the absence and presence of VEGF-A (40 ng/ml). The analysis was conducted 3 h after cell seeding using confocal microscopy (Nikon). The parameters of the tube formation including total loops, total tube length, and total branching points per image were assessed applying Wimasis Image Analysis Service.

Matrigel Growth Factor Reduced Basement Membrane Matrix (22 mg/mL [BD Biosciences]) was diluted to 8.8 mg/mL in cold, sterile PBS, and 100 μL was transferred into each well of a chilled 96-well plate. The plate was incubated for 2 h at 37°C to set the Matrigel. HUVEC cells were washed with PBS, lifted from the flask, and washed again with serum-free basal EGM-2 medium (Lonza). Then, 10,000 cells were added per well together with control medium or medium from MLS 402 cells pretreated with drug. The negative control medium was basal EGM-2 medium (serum-free), and the positive control medium was EGM-2 medium supplemented with growth factors and 2% FCS that were supplied with the medium. The plate was incubated at 37°C in 5% CO₂, and images were recorded at 1 h intervals for 12 h by a ProgRes MF cool camera attached to an Axiovert 40 CFL microscope, using ProgRes Mac Capture software (version 2.8.3). Tube lengths were measured using ImageJ (version 1.47d).

Human pluripotent stem cells, including human embryonic fibroblast-derived hiPSCs (HEF-hiPSCs; designated WT1) and H9 cells (a human embryonic stem cell line; designated WT2) [29 (link)], were maintained on Matrigel Growth Factor-Reduced Basement Membrane Matrix (BD Biosciences, San Diego, CA, USA)-coated 6-well plates in mTeSR1 medium (Stem Cell Technologies, Vancouver, Canada). The culture medium was replaced daily. Cells were passaged every 4–5 days using 0.5 mM EDTA (Invitrogen, Carlsbad, CA, USA) at a split ratio of 1:6.