Axiovert 25 microscope

At 72 h post-transfection the cell cultures were photographed using a Zeiss Axiovert 25 microscope at 5x magnification (Carl Zeiss, Jena, Germany). Following trypsination, cells were counted using Beckman Coulter Z2 (Beckman Coulter, Inc., Fullerton, CA, USA).

The invasion test was performed as described by Martínez-Rodríguez et al. [84 (link)] with some modifications. Briefly, 1.2 × 10³ cells were seeded per well, and after the formation of the spheroid, it was treated with 800 and 1000 μg/mL of CSE and 9.94 μg/mL of cisplatin for 24 h. Subsequently, 100 μL of Matrigel^® Matrix (Corning^®) per well and then RPMI medium was added. The invasion was determined using an inverted Zeiss Axiovert 25 microscope (Carl Zeiss) and ImageJ software and calculated by measuring the area and perimeter between the spheroids’ edge and the invading cells’ edge.

Various cell concentrations were tested to determine an optimum inoculation density that would result in the fastest growth rate within 48 hr of seeding. Bioreactors were inoculated at concentrations of 20, 30, and 60 cells/bead. Cell counts and images were taken at 12 hr, 24 hr, then every 24 hr following for a total of 4 days. For each condition, two bioreactors were inoculated, and two 3-mL samples were taken from each bioreactor at each time point to count. Each 3-mL sample was put into a 15-mL conical tube, and the microcarriers were settled for 5 min. The supernatant was removed and the microcarriers were washed three times with 1 mL of Ca⁺/Mg⁺ free PBS with 1% Anti-Anti solution as described previously. The cells were then re-suspended in 1 mL of PBS, and two 100-μL samples were taken from each conical tube to be counted with the NucleoCounter. Images of the cells on the microcarriers were taken with a Zeiss Axiovert 25 microscope (Carl Zeiss). A 0.5-mL sample was removed from each bioreactor using a 5-mL pipette and added to a 6-well plate with 1.5 mL of PBS. Twenty microliters of 0.5% crystal violet (Sigma Aldrich) in methanol was added to each well and left to sit for 5 min at room temperature. Images were taken at 10× magnification with the filter set to phase 0, as this gives a flatter image that refracts less light making the cells easier to see.⁸ (link)

Transfected or untransfected AGS_GR cells were grown as confluent monolayers in 24-well plates before a cell-free region was created using a 2-μL pipette tip. Cells were washed twice in PBS, then washed twice in serum-free media before the treatment was applied. Whole cells that had migrated into the denuded region were counted and scratch wound width was measured using a graticule at 0 and 8 hours after treatment. Representative images were taken at these times using a Zeiss Axiovert 25 microscope (Carl Zeiss Microscopy).

An aggregate was defined as a group of cells >40 μm in diameter. For both bioreactor and static culture conditions, the aggregate diameter was measured for 60 aggregates (n = 20 per animal, 3 animals for each condition) at the end of each serial passage. Aggregate size was determined by taking multiple 1.0‐ml samples to be imaged using a Zeiss Axiovert 25 microscope (Carl Zeiss Microscopy, Thornwood, NY, http://www.zeiss.com).