Celigo image cytometer system

After infection with lentiviruses expressing sh-MTHFD1L or scrambled shRNA, OSCC cells were cultured for 48 h and harvested during the logarithmic phase. Next, cell suspensions were generated and seeded in triplicate in 96-well plates at a density of 1,000 cells per well. After culturing at 37 °C with 5% CO₂ for an additional 24 h, images of the cell were captured daily for 5 d using a Celigo image cytometer system (Nexcelom; Lawrence, MA, USA). Cell numbers per well were also quantified using the Celigo system, and cell growth curves were plotted for each condition.
The cell viability was assessed using the MTT (Genview, Beijing, China) assay. Briefly, 1,500 shRNA-treated or untreated cells per well were seeded in 96-well plates. After 24 h at 37 °C, MTT (5 mg/mL) was added to each well before the terminal point of cell culture, and cells were incubated for an additional 4 h. Then, the medium was removed and formazan crystals were dissolved in 100 μL dimethyl sulfoxide (DMSO). The cell viability was determined by measuring the absorbance of each well at 490/570 nm and using a multi-well plate reader (Cat. #M2009PR; Tecan Infinite).

TE-1 and KYSE-150 cells were seeded into 96-well plates at a cell density of 2.0×10³ cells/well, with three replicates/group. The cells were subsequently cultured with 100 µl RPMI-1640 medium for a total of 5 days. Every 24 h, a Celigo image cytometer system (Nexcelom Bioscience LLC) was used to determine cell number, and the number of cells with green fluorescence in each well was calculated using a Celigo cell count (14 (link)).