Multiskan spectrum spectrophotometer

Cell sensitivity to PTX was examined by MTT assay. Briefly, cells were seeded in the 96-well plate at a density of 4.5 × 10³ cells per well and incubated overnight. After that, the medium was changed to fresh medium containing various concentrations of PTX. After 72 h, 20 μl MTT was added to each well and cells were incubated for 4 h with MTT at the concentration of 500 mg/ml. Then, the precipitate was dissolved completely by 150 μl DMSO and the light absorbance was measured at 570 nm using the Multiskan Spectrum spectrophotometer (BioTek Instruments, Inc. Vermont, USA).

For the migration assay, 200 µL of TPC-1 or SW-579 cells at a density of 2 × 10⁵ cells/mL in serum-free culture medium containing PVE at 4, 2, 1, or 0 mg/mL was seeded on upper migration chambers (Corning Incorporated Costar, USA) in 24-well plates. Six hundred microlitres of drug-free culture medium containing 10% v/v FBS was added to the lower chamber of each well. After incubation for 36 to 48 h, the cells were fixed with methanol and stained with 0.1% m/v crystal violet. Cells that had not migrated to the upper chamber were gently scraped away using cotton swabs. Then, 33% v/v acetic acid (100 μL) was used to elute crystal violet, and the OD value at 490 nm was detected with a Multiskan Spectrum spectrophotometer (BioTek Instruments), and cells that had migrated to the upper chamber were quantified.

Nthy-ori 3–1, TPC-1, FTC-133, SW579, and BCPAP cells were seeded at a density of 5 × 10⁴ cells/well in 96-well plates. After 12 h, the original medium was exchanged with a PVE solution (1 mg/mL) diluted with complete culture medium. Then, they are continued to culture for 24 h, 48 h, and 72 h. And, 20 μL of an MTT (5 mg/mL, Solarbio) solution in PBS was added into each well, respectively, and cells were continually incubated for another 4 h. Then, 150 μL of DMSO (Solarbio) was added to each well, and cells were incubated with shaking at 37°C for 8 min. The absorbance of each well at 490 nm was determined using a Multiskan Spectrum spectrophotometer (BioTek Instruments, USA). Each experiment was performed in triplicate and repeated at least three times.