Prism excel

Cells were treated with vitamin B2 0.125 μg/mL, B6 125 μg/mL and 250 μg/mL or B9 125 μg/mL, (corresponding with IC-50 amounts for 3 days after which fresh media and vitamin B added and allowed to incubate a further 3 days. Controls included untreated and NaOH at corresponding doses to the B vitamins dissolved in NaOH. Cell migration was performed using the Boyden chamber assay 8-µm pore size membrane filter inserts in 24-well tissue culture plates. The cells were trypsinized and re-suspended in serum-free RPMI-1640 media at the density of 2 × 10⁵ cells/mL. A total of 200 µL of cells suspension was seeded in the upper chamber of the trans wells, and 600 µL of media into the lower chamber. The chambers were incubated at 37 °C in a humid atmosphere with 5% CO₂. After 20–22 h, the non-migrating cells on the upper surface of the insert were removed and the cells that migrated to the underside of the membrane were counted using a light microscope. Excel and Prism excel (Graph Pad Software, La Jolla, CA, USA) were used to aid in the statistical analysis using Student’s t-test and p < 0.05 was considered significant.