12 well transwell chamber

The 12-well transwell chamber (8 µm pore size; Corning, NY, USA) was applied to analyze the migratory and invasive abilities of pancreatic cancer cells. For the transwell cell migration assay, 2 × 10⁵ pancreatic cancer cells in 200 µl serum-free DMEM were directly seeded into the upper chamber, while 800 µl DMEM with 10% FBS was added to the lower chamber. After 24 h incubation, the migrating cells were fixed with 4% paraformaldehyde and stained with crystal violet. For the transwell cell invasion assay, the 12-well transwell chamber was coated with 50 µl of 1:8 mixture of Matrigel (BD Biosciences) and serum-free DMEM for 2 h at 37 ℃. Then the 4 × 10⁵/ml pancreatic cancer cells were seeded into the upper chamber and 800 µl DMEM containing 10% FBS was added to the lower chamber. After 24 h, the cell was fixed and stained followed by imaging at 100 × magnification under an inverted light microscope. The number of cells that migrated and invaded through the chamber filter was counted by the ImageJ software.

Scratch‐wound assay was performed to measure the cell migration speed. Approximately 2 × 10⁵ HUVECs and 2 × 10⁵ HaCaT cells were inoculated onto 12‐well plates and cultured to 90% confluency. First, the cells were cocultured with 250 μM H₂O₂ for 4 h, and a straight‐line scratch was created using a 200‐μL pipette tip. Then, each group was subjected to different treatments, and photographs were collected under a microscope at predetermined timepoints.
A 12‐well Transwell chamber (Corning, USA) with 8‐μm pore size was used to observe the migration of HUVECs. Approximately 3 × 10⁵ cells were inoculated onto the upper chamber containing serum‐free DMEM, and DMEM containing 10% FBS was used in the lower chamber; the cells were treated with H₂O₂. Next, 100 μL PBS, 200 μg PEG–PPS, and 2.6 μg LL‐37 and LL‐37@PEG–PPS (containing 2.6 μg LL‐37 and 197 μg PEG–PPS) were added into the lower chamber and then cocultured for 24 h. The cells that passed through the pores were fixed with methanol, stained with 1% crystal violet, and enumerated.

The effect of dystroglycan depletion on Kasumi-1 differentiated cells on migration was evaluated using a 12-well Transwell chamber (Corning, Costar Corp Cambridge, MA, USA). Kasumi-1 differentiated cells were added to the upper chambers and allowed to migrate for 120 min at 37°C in 5% CO₂ towards the lower chamber containing 10⁻⁷ LPS. Migrated cells from lower chamber were centrifuged at 1200 rpm for 5 min, collected at the bottom of the lower chambers and quantified; the result was expressed as a percentage of cells added to the top of the plate.