Model ckx41

Neurospheres were generated from isolated NPCs in the hippocampal area of postnatal 24-hour-old rat. Briefly, rat brains were coronally sectioned, and the hippocampal area was obtained, which was followed by tissue and cellular dissociation. Isolated cells were cultured at a density of 5 × 10⁵ cells/mL in DMEM-F12 proliferation medium containing 2% B27 supplement, 20 ng/mL bFGF, and 20 ng/mL EGF. The cultures were observed and photographed daily under a phase contrast microscope (Model CKX41, Olympus, Japan). After 2 passages, cells were cultured for 72 and 96 h, Olympus imaging analysis system was employed to observe of neurospheres, and the number and diameter of neurospheres were counted and measured separately in 10 randomly selected microscopic fields (100x) for each flask.

Normal‐type PC12 cell line was purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). Meanwhile, wide‐type and Ras mutant‐type PC12 cells (RasN17) were provided by Professor Hiroyuki Osada.11 In NGF‐mimic assay, 20 000 PC12 cells were seeded in each well of a 24‐well microplate in 1 mL CM medium (Dulbeccor's modified Eagler's medium [DMEM; Thermo Scientific] containing 10% foetal bovine serum, 5% horse serum and 1% pre‐mixed antibiotics [Invitrogen]) and incubated in 5% CO₂ at 37°C. The medium was replaced after 24 hours with 1 mL serum‐free DMEM medium containing a test sample or 0.5% dimethyl sulphoxide (DMSO). In the inhibitor test, the cells in 24‐well microplates were pre‐cultured with 500 μL medium containing an inhibitor for 30 minutes. Then, another 500 μL medium containing a test sample or 0.5% DMSO was added. The inhibitors of TrkA, ERK, GR, PLC, PKC and Ras (K252a, U0126, RU486, U73343, GO6983 and S3131) were obtained from Sigma. The Raf and TrkB inhibitors (AZ628 and ANA‐12) were purchased from Axon Medchem BV and Selleckchem, respectively. After 48 hours, the morphological changes in the cells were observed using a phase‐contrast microscope (Model CKX41; Olympus). About 100 cells were counted in each of three randomly chosen fields. A positive cell was defined as the neurite outgrowth of a cell longer than the diameter of cell body.

A 48-well plate coated with EC matrix and solidified at 37°C was taken and 2 x 10⁴ HUVEC cells (Human umbilical vein endothelial cells) were seeded in each well containing 300 μL of endothelial growth media and allowed to grow for 6 h. Cisplatin, Etoposide or C-10 were added to cells and incubated for 24 h. Then the cells were observed under inverted microscope (Model-CKX41, Olympus) to analyse the tube formation and images were obtained at 4x magnification.

Transwell chambers were placed with 30 μl of diluted Matrigel in a 24-well plate and incubated at 37°C for 2 hrs. 1×10⁵ MDAMB-231 cells in serum-free DMEM were seeded into the Matrigel of Transwell chambers. 500 μl of DMEM with 10% FBS was added to the basal chamber and incubated at 37°C with 5% CO₂for 48h.Cells were fixed by replacing the culture medium in the bottom and top of the chamber with 4% formaldehyde dissolved in PBS. After fixation for 15 min at room temperature, the chambers were rinsed in PBS andpermeabilized with methanol. The chamber is stained with 0.2% crystal violet for 10 min. Excess stain was washed away with dH₂O. The cells were visualized under inverted microscope (Model CKX41, Olympus).

For a graphical overview of the time course of the different protocols and their respective steps, see Figure 6A. The IPEC-J2 cells (ACC 701, DSMZ, Braunschweig, Germany) were cultured in growth medium (see above) at 37 °C and 5% CO₂ [18 (link)]. The culture medium was refreshed daily. Approximately 1 × 10⁴ cells were plated per well in a 96-well plate (25-μm flat film bottom, Eppendorf) and grown for 3 days when 40–50% confluence was attained. Cell plates were imaged under a light microscope (Model CKX 41, Olympus, Tokyo, Japan) combined with a digital color camera (Model UC 30, Olympus, Tokyo, Japan). The Olympus cellSens Standard software (Olympus, Tokyo, Japan) was used to capture the images without any further processing. Passage (passage number 4–5), number of seeded cells and growing period were standardized in order to achieve a homogeneous cell counting prior to the in vitro test.