Neutrophil Collagenase

Primary chicken preadipocytes (PCPs) were isolated from the abdominal adipose tissue of 10-day-old AA broiler chickens as previously described [36 (link)]. Briefly, chicken adipose tissue was collected and washed with PBS, and visible blood vessels were removed. The adipose tissue was minced into sections of approximately 1 mm² with scissors and incubated with 10 ml of digestion buffer (DMEM/F12, 100 mM HEPES, 1.5% BSA, pH 7.4) supplemented with 2 mg/ml collagenase (Type 1, Invitrogen) with shaking for 65 min at 37°C. Flask contents were mixed and filtrated through nylon screens with 100- and 25-μm mesh openings to remove undigested tissue and large cell aggregates. The filtered cells were centrifuged at 300 x g for 10 min to separate the floating adipocytes from the pellet of stromal-vascular cells. The stromal-vascular cells were then resuspended with erythrocyte lysis buffer, incubated at room temperature for 10 min and then centrifuged at 300 x g for 10 min. The stromal-vascular cells (including the preadipocytes) were washed with DMEM/F12 and seeded at a density of 5×10⁴ cells/ml in DMEM/F12 media supplemented with 10% FBS plus 100 units/ml penicillin and 100 μg/ml streptomycin, at 37°C with 5% CO₂. The PCPs were serially subcultured at a 1:2 split ratio until senescence. The following formula was used to determine the population doublings of each subculture and to establish the PD growth curve: PD = ln(N_finish/N_start)/ln2, where PD is the number of population doublings, ln is the natural logarithm, N_start is the number of cells initially seeded, and N_finish is the total number of cells recovered at subculture.
GP2-293 cells, NIH 3T3 cells, DF-1 cells, and HEK-293 cells were grown in DMEM supplemented with 10% FBS plus 100 units/ml penicillin and 100 μg/ml streptomycin, at 37°C with 5% CO₂.

Directly after sacrifice, pancreata of one-month old mice were injected with 2 ml Collagenase P solution (1.33 mg/ml Collagenase P (Roche) in HBSS (Gibco)), cut out, minced with a scalpel and gently shaken for 30 min at 37 °C in 5 ml Collagenase P solution. All subsequent steps were performed at 4 °C in a laminar flow cabinet and all centrifugation steps were carried out for 3 min at 180 x g. Cells were resuspended in 10 ml 5% FBS in HBSS and incubated 10 min for sedimentation of the cellular fraction. Supernatant was aspirated carefully, and the pellet was washed 3 times with 5% FBS in HBSS. Cells in 10 ml 5% FBS in HBSS were transferred into a new tube through a 100 µm cell strainer, slowly laid over 20 ml 30% FBS in HBSS and centrifuged. Cells were resuspended in 2 ml recovery medium (acinar cell medium, see below, with 30% FCS), incubated at 37 °C for 1 h, centrifuged and resuspended in a 1:1 mixture of acinar cell medium (containing 0.1% bovine serum albumin, 0.2 mg/ml soybean trypsin inhibitor (Sigma), 1% ITS premix (Corning), 50 µg/ml bovine pituitary extract (ThermoFisher), 0.1% FBS, 0.5% penicillin/streptomycin, 0.25 µg/ml Fungizone antimycotic (ThermoFisher) in Waymouth’s medium (Gibco) and rat tail collagen type I (Corning). Per pancreas, cells were seeded into 16 wells of a 48-well plate on a previously prepared collagen layer (final collagen concentration 2.5 mg/ml) and covered with another collagen layer before adding acinar cell medium. Medium was changed every 24 h.
Five days after seeding, images were acquired with AxioVision Rel 4.8 software and the percentage of ductal structures of the total amount of acinar explants was determined by counting 5 microscopic fields of view at 100x magnification for each pancreas. Quantification was blinded to the genotype.

Satellite cells (SCs) were sorted as previously reported (Ryall et al., 2015 (link)). Briefly, hindlimb muscles from 6-week-old male mice were harvested, and blood, tendons, lipid tissues and nerves were removed. The muscles were finely chopped until a slurry was generated. The slurry was digested with 0.2% collagenase II and 0.1% dispase at 37°C for 60 min. The slurry was centrifuged, filtered and incubated with a primary antibody cocktail mixture as follows: APC-labeled α7-integrin, PE-labeled CD11b, CD31, CD45, and Sca1 and 7-AAD. Sorting was conducted on SH800S (SONY) and the gating strategy was based on positive α7-integrin, negative 7-AAD and CD11b/CD31/CD45/Sca1 staining.