3d matrigel matrix

In vitro HUVEC tube formation assay was conducted in a tumor cell environment by co-incubation and 3D Matrigel Matrix (Corning). The co-incubation was carried out in a transwell system (12-well plate). The CT26 cells were cultured on the upper insert, and the HUVEC were seeded in the plate with Matrigel Matrix (1%, w/v). Then the RPMI-1640 medium (FBS 10%) with free RG, M-γ-CD, Mix, RG@γ-CD, and RG@M-γ-CD were added, and the cells were co-incubated in 37 °C and 5% CO₂ for 6 h. The endothelial cell tube formation was observed by fluorescence microscope (Carl Zeiss) in bright field. In addition, HUVEC were seeded with Matrigel Matrix (0.5%, w/v) in a six-well plate, then were treated as above. The treated HUVECs were washed and cultured in serum medium containing Matrigel Matrix for 2 h. Then the cells were collected and treated with RIPA lysate (Beyotime) for phosphorylation examination.

TNBC tissues were cut into small pieces and mechanically homogenised. The homogenised tissue was digested with trypsin on ice. After centrifugation, single‐cell suspensions were added dropwise with cold 3D Matrigel matrix (Corning, 354348) to six‐well culture plates, which were kept inverted in a cell incubator for 30 min before organoid‐specific medium was added. Organoids were cultured with media containing 1% GlutaMax (Thermo Fish, 35050061), 100 μg/mL Primocin (InvivoGen, 26‐69‐PM), .5 μg/mL hydrocortisone (MedChemExpress, HY‐N0583), 10 μM Y27632 (Sigma, Y0503), .2 nM Wnt3a (StemRD, W3a‐H‐025), 250 ng/mL R‐spondin1 (PeproTech, 120‐38), 100 ng/mL Noggin (PeproTech, 120‐10D), 1× B27 + Vitamin A (Invitrogen, 17504‐044), 10 mM HEPES (Thermo Fish, 15630080), 20 ng/mL Fibroblast Growth Factor‐10 (FGF‐10) (Peprotech, 100‐26), 5 ng/mL Epidermal Growth Factor (EGF) (Peprotech, GMP100‐15), 10 mM nicotinamide (Selleckchem, S1899), 100 nM β‐estradiol (Sigma, E2758), 10 μM Forskolin (MedChemExpress, HY‐15371), 5 nM Heregulin B1 (ProSpec, CYT‐733), .5 μM A83‐01 (MedChemExpress, HY‐10432), .5 μM SB202190 (Selleckchem, S10077), 1.25 mM N‐acetylcysteine (Sigma, A0737) and Dulbecco's Modified Eagle Medium (DMEM).