Round bottomed ultra low attachment 96 well plates

Non-transfected or transfected PANC-1 cells grown under either normal or acid adaptation conditions were formed into spheroids in their respective medium or in normal pH conditions following acid adaptation to simulate the 2D model of acid recovery conditions. In total, 2000 PANC-1 cells were seeded in round-bottomed, ultralow attachment 96-well plates (Corning, NY, USA) in 200 μL growth medium, supplemented with 2% GelTrex LDEV-Free reduced growth factor basement membrane matrix (Thermo Fisher Scientific, Waltham, MA, USA). After seeding, cells were spun down at 750 RCF for 20 min and were grown for 9 days at 37 °C with 95% humidity and 5% CO₂, and 100 μL of the respective pH medium was exchanged every second day. Light microscopic images of the spheroids at 10× magnification were acquired on days 2, 4, 7, and 9. On day 9, PANC-1 spheroids, either non-transfected or transfected cells (in replicates of three) were transferred to a black 96-well plate with 100 µL of the respective pH medium and 100 µL CellTiter-Glo^® 3D Reagent (Promega, Madison, WI, USA). This plate was wrapped in aluminum foil, shaken for 5 min, and then incubated without shaking for 25 min at room temperature. The luminescence signal was recorded using a FLUOStar Optima microplate reader (BMG Labtech, Ortenberg, Germany).

PDAC cell lines were first suspended in normal culturing medium with a final concentration of 200 cells per 200 μL. The resuspended cells were then seeded onto ultra-low attachment 96-well round-bottomed plates (Corning Inc.), which were then left untouched for 4 days. On day 4, 100 μL of the culturing medium from each well was carefully removed and replaced with 100 μL of 30% Matrigel (BD Bioscience) in normal medium, with or without Gem. Spheroid growth was monitored and photographed at 10 × magnification using an inverted Axiovert microscope (Zeiss) every day for up to 5 days, and the spheroid diameters were measured using ImageJ software. For conditioned medium experiments, 100 μL of the culturing medium was replaced with 30% Matrigel in 100 μL serum free conditioned medium derived from placebo or Gem-treated PSCs, together with or without Gem on day 4 instead. For invasion measurement with DT6066/TB32048 derived parental control and GemR spheroids, the relative invasion was determined from images of each spheroid using ImageJ by subtracting the solid spheroid area from the maximal invaded spheroid area and normalizing to the control.

For spheroid generation, 100 μl/well of cell suspensions at 1 x 10⁵ cells/ml were dispensed into ultra-low attachment 96-well round-bottomed plates (Corning B.V. Life Sciences, Amsterdam, The Netherlands) using a multichannel pipette. Plates were incubated for 24 h at 37° C under 5% CO₂. Images were captured using a microscope (Nikon) equipped with a CCD camera and imported into HKBasic software.

The C2C12 mouse skeletal myoblasts were obtained from Pythonbio (Guangzhou, China). The cells were grown in DMEM supplemented with 10% FBS and 1% P/S. At 70–80% confluence, the cells were cultured in DMEM supplemented with 2% horse serum and 1% penicillin-streptomycin for six days to induce differentiation into the myotubes [32 (link)]. All the cells were maintained at 37 °C in a humidified atmosphere containing 5% CO₂. The culture media were replaced every two days.
After differentiation, the cells were seeded at 1 × 10⁴ cells per well into black-bottom 96-well plates (Corning, Kennebunk, ME, USA) overnight for the 2D culture. The cells were seeded at 2000–3000 cells/well into ultra-low attachment 96-well round-bottomed plates (Corning, Kennebunk, ME, USA) and then cultured for three days to generate skeletal muscle spheroids (3D culture) [33 (link)]. The cells were then treated with varying concentrations of kaempferol (50, 25, 12.5, and 6.25 μM) for 24 h in the 2D culture and 48 h in the 3D culture. The treatment concentration of kaempferol was determined based on our previous cell activity experiments. The cells were cultured at 37 °C in a humidified atmosphere containing 5% CO₂. The testing was subsequently commenced.

For spheroid generation, 5 × 10² cells were seeded into ultra–low attachment 96 well round–bottomed plates (Corning, New York, NY, USA) containing sphere–forming medium DMEM/F12 (Invitrogen, Waltham, MA, USA) supplemented with 1× B27 serum substitute (Invitrogen), 20 ng/mL of mouse recombinant epidermal growth factor (PeproTech, Cranbury, NJ, USA), and 10 ng/mL of basic fibroblast growth factor (PeproTech). The DFX and DFO treatment groups were exposed to 20 μmol/L of DFX and 50 μmol/L of DFO, respectively. After 6 days of using Image J, the length of the radius of spheroids was examined. The volume (μm³) of each spheroid was calculated using the following formula: V = πR³4/3.