Collagen Contraction Assay for Fibroblasts

The collagen contraction assay was performed as previously described^{13 (link),35 (link)}. Fibroblasts were seeded in T25 flasks at 5 × 10⁵ cells per flask with 5 mL of normal growth media. Twenty four hours after seeding, the cells were either sham irradiated or given 2 Gy of X-rays. Cells were harvested 48 h after treatment and resuspended at 4.5 × 10⁵ cells per 100 μL of FBS. Rat tail collagen (Corning, cat. 354249, Corning, NY, USA) was diluted in 0.5 M glacial acetic acid and 10X DMEM to a final concentration of 2 mg/mL. Cells were embedded in the collagen at 1.5 × 10⁵ cells per 500 μL in a low attachment 24-well plate. Once the collagen discs were solidified, the collagen was released from the sides of the well, and suspended on 500 μL complete growth media. After 5 h of contraction, the discs were imaged and the area of the collagen discs were measured using ImageJ software.