Ts100 inverted microscope

Pre-formed alginate hydrogel microtubes were imaged using an inverted microscope (Nikon Eclipse TS100, Micro Video Instruments, Avon, MA, USA) equipped with a Retiga 2000R digital camera and QImaging software. Optical images of microtubes were analyzed using NIS-Elements D software, and inner and outer diameters of the microtubes were calculated as the mean ± standard deviation. The wall thickness was calculated by subtracting the inner diameter from outer diameter and dividing by 2. Cell growth in hydrogel microtubes was monitored daily using the Nikon TS100 inverted microscope. Optical images were captured using a 4x objective lens on days 1, 4, and 7.

Human neuroblastoma cell lines SK-N-AS, SK-N-DZ, and SHEP1 were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS, Thermo Fisher Scientific, Waltham, MA, USA). Media were also supplemented with antibiotics (penicillin and streptomycin) at a 1% concentration (P/S, Thermo Fisher Scientific, Waltham, MA, USA). BE(2)-C cells were cultured in a 1:1 mixture of DMEM and Ham’s nutrient mixture F12 (DMEM/F12, Thermo Fisher Scientific, Waltham, MA, USA) that was also supplemented with 10% FBS and 1% P/S antibiotics. All four cell lines were purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA). The lentiviral packaging cell line 293FT was cultured in DMEM containing 10% FBS, 1% P/S, 0.5 mg/mL G418, 4 mM L-glutamine, 0.1 mM MEM non-essential amino acids, and 1 mM MEM sodium pyruvate. The 293FT cell line, growth media, FBS, and supplemental reagents were obtained from Thermo Fisher Scientific (Waltham, MA, USA). All cells were cultured in a fully humidified incubator at 37 °C with a 5% CO₂ atmosphere. Cells were photographed using a Nikon TS100 inverted microscope (Nikon Corporation, Tokyo, Japan) at 40× magnification. The Image-Pro Plus 6.0 software program (Media Cybernetics, Inc., Rockville, MD, USA) was used for micrograph analyses.

Live motility experiments were performed in 12-well plates (range of 20,000–50,000 cells/well depending on the cell culture used), in which cells were treated with TSA (1 μM), SAHA (2 μM), IFN-γ (1 μg/mL), or their combination, the day after seeding. To measure cell migration, 6 different areas per well were randomly recorded overnight (~16 h), to achieve a time-lapse monitoring of cell movements with an Axio Observer microscope (Zeiss, Jena, Germany) with controlled atmosphere and temperature. Images were captured every 30 min during a 16 h timespan and then cellular motility was analyzed by tracing single cell coordinates (x, y) with the MTrackJ plugin of ImageJ (https://imagej.nih.gov; accessed on 31 January 2020).
To measure cell ability to close a wound during scratch assays, GBM cells (HuTuP61, HuTuP82 and HuTuP176) were plated onto 12-well plates at high confluence (100,000–150,000 cells/well depending on the primary culture), and, after 24 h, the cell monolayer was scratched and treated or not with HDI (1 μM TSA and 2 μM SAHA). Then, the ability of edge cells to move into and close the scratch during time (24 and 48 h) was measured. Images were acquired with a Nikon TS100 inverted microscope (Nikon, Melville, NY, USA) and wound width was measured in four random fields/well using Adobe Photoshop CS6 (Adobe Systems Incorporated, La Jolla, CA, USA).