Collagenase type xi s

Tissue samples (~0.25 cm²) were harvested from the ear of adult (6 mo) control and DEX-exposed mice under terminal anesthesia. The tissue was rinsed in Hank’s Balanced Salt Solution (HBSS) (Life Technologies Europe BV, Stockholm, Sweden), then minced with sterile razor blade into Collagenase (Type XI-S) (Sigma-Aldrich, Sweden) (30 min at 37 °C). After digestion, 3 mL of DMEM Medium (Life Technologies) supplemented with 10% Fetal Bovine Serum and 1% Penicillin/Streptomycin (Life Technologies) was added to a 6 cm plate and the samples were incubated at 37 °C for at least 6 days. After passaging (0.05% Trypsin-EDTA; Invitrogen), the cells were plated in 35 mm dishes in MEF medium (DMEM Medium +10% FBS +1% pen/strep) at a density of at least 50 k/cm². After 24 h, the expression of clock genes was synchronized by exposing the fibroblasts to 1 µM DEX. The cells were collected between 36 and 63 h after synchronization. The relative expression of Bmal1 was assessed by qPCR with GAPDH as housekeeping gene. Circadian oscillations in clock gene expression were analyzed by means of cosinor rhythmometry^{52 ,53 (link)}.

Tissue samples (~0.25 cm²) were harvested from the ear of adult (6 mo) control and Dex-exposed mice under terminal anesthesia. The tissue was rinsed in Hank's balanced salt solution (Life Technologies Europe, Stockholm, Sweden), then minced with sterile razor blade into Collagenase (Type XI-S) (Sigma-Aldrich) (30 min at 37 °C). After digestion, 3 ml of Dulbecco's modified Eagle's medium (DMEM; Life Technologies Europe) supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin (Life Technologies Europe) was added to a 6-cm plate and the samples were incubated at 37 °C for at least 6 days. After passaging (0.05% Trypsin-EDTA; Invitrogen, Life Technologies Europe), the cells were plated in 12 multi-well plates in MEF medium (DMEM medium+10% fetal bovine serum+1% pen/strep) at a density of at least 50k cm⁻². After 24 h, the expression of clock genes was synchronized by exposing the fibroblasts to 1μm Dex. The cells were collected between 6 and 36 h after synchronization. The relative expression of Bmal1 was assessed by quantitative PCR with Gapdh as the housekeeping gene (see also Supplementary Materials and Methods). Circadian oscillations in clock gene expression were analyzed by means of cosinor rhythmometry.^{38 (link), 39 (link)}