Cell Migration Assay with IL-4 and IL-13

The 70 µL of cell suspension containing 5 × 10⁴ cells was applied into each well of the Culture-Insert 2 Well (placed in 24-well cell culture plate; Ibidi, Gräfelfing, Germany) and cultured for 24 h. Subsequently, the inserts were removed from wells and wells were rinsed with DPBS, followed by the addition of medium without or with 100 ng/mL of IL-4 or IL-13. The closure of insert-created gap (wound) was observed in three (Caco-2) or four (HCT 116) independent experiments after 24, 48, and 72 h under the CKX41 inverted microscope with SC30 camera (Olympus, Tokyo, Japan). For HT-29 cells, the experiment was conducted up-to 144 h and exclusively for IL-4. Gap area was measured using the MRI Wound Healing Tool macro for ImageJ software (NIH) [69 (link)]. Subsequently, the ratios of measured gap areas were calculated (area at a given time point/area at time 0) and statistically compared between stimulated and unstimulated cells. Additionally, mean gap areas (expressed as a percent of gap area at time 0 h) were plotted against time.