Isolation of mouse fibroblasts

Fibroblasts were isolated from the ears and tails of male C57BL/6 mice according to a published procedure³⁴. In brief, mice were euthanized by exposure to CO₂ and cervical dislocation, after which ears and 5 cm of tail were sterilized in 70% ethanol for 5 minutes. After air drying, tissues were cut into small, ~3 mm pieces and digested with a mixture of collagenase and pronase for 90 minutes at 37°C with gentle shaking. Following enzymatic digestion, the tissues were ground in 70 μm cell strainers using a 10 ml syringe plunger, and the resulting cell suspension was centrifuged at 500xg for 5 minutes at room temperature. The cell pellet was resuspended in fresh fibroblast culture media (DMEM/F12, 10% FBS, 1X antibiotic-antimycotic, 50 μM β-mercaptoethanol, 1X non-essential amino acids), spun once more at 500xg, resuspended in 10 ml fibroblast culture media, plated in 10 cm tissue culture dishes, and incubated for 3 days at 37°C, 5% CO₂, and 3% O₂. Following 3 days of incubation, media was replaced to get rid of cellular debris, and the cells were then incubated further until reaching ~80% confluency. Upon reaching the desired confluency, ear and tail fibroblasts were trypsinized and frozen at 1 million cells per cryovial in fibroblast freezing media (fibroblast culture media with 50% FBS and 10% DMSO).