Eclipse ts100 light microscope

P815-NT, P815-KD1 and P815-KD2 (1 × 10⁵) cells were plated in complete DMEM in triplicate wells, and total cell numbers were scored at 24, 48 and 72 hours using an automated cell counter (Beckman Coulter Z2 particle analyzer). For colony formation assays, P815-NT and P815-KD2 (2 × 10⁴) cells were suspended in MethoCult media (M3231; Stem Cell Technologies) and plated in a 35 mm sterile tissue culture plate as per manufacturer's instructions. Colonies were manually counted after 8 days by phase contrast microscopy (Nikon Eclipse TS 100 light microscope). For inhibitor studies, HMC-1 or P815 (2 × 10⁴) cell were plated with DMSO or II-B08 with indicated concentrations with further addition of same doses at 72 h to maintain inhibitor concentration throughout experiments. Images were acquired and colonies were counted at day 8. P815-NT and P815-KD2 were incubated with DMSO or SU11652 (250 nM) for 18 hours, stained with FITC-AnnexinV and propidium iodide (PI). Apoptotic cells were identified as AnnexinV⁺PI⁻ cells by flow cytometry (gating was based on unstained cells).

To study cell proliferation effects in a 3-dimensional model, cells were seeded into the inner wells non-coated 96-well microtiter plates (5 × 10⁴ cells/mL) in 0.1 mL/well of media containing N2 (1% v/v), EGF (10 ng/mL) and bFGF (10 ng/mL) at 37 °C/5% CO₂. Outer wells were filled with sterile PBS (0.1 mL/well). Treatments were added after 24 h as previously described and proliferation was monitored daily using an MTT assay and visually by a microscope. Data were normalized to the presence or absence of EGF + bFGF (EGF + bFGF and EGF/bFGF-free medium were set at 100% and 0%, respectively). In vitro cell proliferation was also studied by analysis of neurosphere number and mean diameter. Pictures of the wells of a 96-well plate treated as described above were taken immediately after addition of treatments (t = 0) and daily thereafter using a Canon PC1234 digital camera, (Japan) attached to a Nikon Eclipse TS100 light microscope (UK). The number of neurospheres and their average diameter was screened using random sampling (1 visual field per well, n = 6) and quantified using ImageJ software (Rasband, 1997–2015 ). Neurospheres were classed based on their mean diameter: small (50–100 μm), medium (100–200 μm) and large (≥200 μm). Neurospheres on the edges of each image were excluded from analysis, so that only completely visualized spheres were quantified.

For purifying 100,000–150,000 nuclei, 1 g of fresh tissue was harvested and ground in liquid nitrogen using a mortar and pestle to break the cell wall. To avoid enzymatic activity, all the following steps were performed at 4 °C. The resulting powder was transferred into 20 mL NPB (Nuclear purification Buffer containing 20 mM MOPS, 40 mM NaCl, 90 mM KCl, 2 mM EDTA pH 8, 0.5 mM EGTA, 0.2 mM Spermine, 0.5 mM Spermidine and protease inhibitor × 1 (Sigma P2714)) and incubated on a rotator at 4 °C for 10 min. The sample was then transferred through a 40-µm mesh and centrifuged for 10 min at 1000 g and 4 °C. The pellet was gently resuspended in 7 mL NPBt (NPB supplemented with 0.1% Triton X-100). Nuclei were then bound to 25 µl of streptavidin-coated Dynabeads (Invitrogen, M-280 Strepavidin) according to the manufacturer’s protocol and washed with NPB. After washing 4–5 times with 1 mL NPBt, the nuclei were resuspended in 1 mL NPB. To count nuclei, a 20-µl suspension was loaded to a Marienfeld hemocytometer (Neubauer-improved, chamber depth of 0.1 mm, 0630010), and visualized with a Nikon Eclipse TS100 light microscope.

Master 3D Petri dish 35-well arrays (Microtissues Inc., Providence, RI, USA) were used to create micro-wells made up of 2% UltraPureTM agarose hydrogel (Invitrogen, Carlsbad, CA, USA). Micro-wells were seeded with 10,000 cells/well. Cells used in this assay were PANC-1, BxPC-3, AsPC-1, HUVECs, and patient-derived pancreatic adenocarcinoma cancer fibroblast cells. After seeding, 1 mL of the appropriate medium was added, and templates were incubated at 37 °C with 5% CO₂ for 24 h up to 10 days. For hybrid spheroid experiments, pancreatic cancer cells were co-cultured with HUVECs and patient-derived pancreatic cancer fibroblast cells in the ratios: 3:1:1, respectively. Spheroids were formed using the 3D Petri dish templates as previously detailed. Since HUVECs are more sensitive to their environment compared with the other cells used in these experiments, and require an enriched medium for their growth, we used their optimized medium, as previously mentioned, for the co-cultured experiments. Light microscopy images were taken using Nikon Eclipse TS100 light microscope, x4 lens.

NALM6 cell line was plated at cell density of 1 × 10⁶ cells/mL onto the ECM Select Array Kit (Advanced Biomatrix) containing 36 different single ECM protein or combinations, each in 9 micro-spots. Twenty-four hours incubation, the ECM Select Array Kit was washed with PBS to remove non-specifically bound cells and was imaged at 4x magnification using an Eclipse TS100 light microscope (Nikon, Melville, NY).