70 μm filter

Animals were sacrificed using CO₂ inhalation. BAL was obtained by flushing the lungs once with 1 ml PBS via the trachea using a 22G indwelling cannula (Braun, Germany). Following perfusion, the lungs were excised, and the tissue was mechanically homogenized in 1 ml PBS by passing through a 70 μm filter (Corning Inc., USA). Cardiac blood was collected and diluted in PBS. Bacterial CFU were determined by plating serial dilutions of BAL, post-lavage lung homogenates and blood on blood agar plates. The plates were incubated for 16–18 h at 37°C and 5% CO₂, CFU were manually counted and the CFU per ml were calculated.

Subcutaneous adipose tissue from healthy donor sites from patients who received flap repair were collected. The isolation and culture of ADSCs was according to the previous protocol in laboratory [45 (link)]. In brief, the fresh adipose tissue was cut into pieces and digested with collagenase type I (Sigma, USA). The cell precipitation was re-suspended and then filtered with a 70 μm filter (Corning, USA). After re-centrifugation, the cell precipitate was re-suspended in the Dulbecco’s modified Eagle’s medium (DMEM, Gibco, USA) consisting of 10% fetal bovine serum (FBS, Serapro, USA). The ADSCs were passaged 3 to 8 times for experiments.
Human umbilical vein endothelial cells (HUVECs) (#GDC166, CCTCC) and C2C12 cells (#GDC175, CCTCC) were purchased from the China Center for Type Culture Collection (CCTCC, Wuhan, China). HUVECs and C2C12 cells were cultured in culture medium supplemented with 10% fetal bovine serum, 100 μg/mL of streptomycin, and 100 U/mL of penicillin in a 5% CO₂ humidified atmosphere.

Mice were maintained according to the guidelines of the Canadian Council on Animal Care. All protocols were approved by the University of British Columbia Animal Care Committee. Islets were isolated as previously described [30 (link),69 (link)]. Briefly, pancreata from both male and female mice were perfused with 1000U/ml Collagenase XI (Sigma-Aldrich) and incubated for 15 minutes at 37°C. Pancreata were manually disrupted and passed through a 70μM filter (Corning) and islets were hand-picked. Isolated islets were allowed to recover for 4–6 hours prior to use in experiments. Islets were cultured in RPMI 1640 supplemented with 10% FBS, 50U/ml Penicillin/Streptomycin and 2mM L-Glutamine at 37° in a 5% CO₂ humidified incubator with or without 1.6ng/ml IFNγ, 0.25ng/ml Il-1β and 0.16ng/ml TNFα (eBioscience) as indicated.

Adipose tissue was harvested from female surgical patients at the University Hospital, Kyoto Prefectural University of Medicine, Japan. Patients gave informed consent, and procedures were approved by the Institutional Ethics Committee for Clinical Research at Kyoto Prefectural University of Medicine (ERB-C-487-1). A previously described protocol was used for isolation and culture of ADSCs, with a slight modification [4 (link)]. Briefly, subcutaneous human fat was enzymatically dissociated at 37°C for 60 min using 0.15% (w/v) collagenase type I (Sigma-Aldrich, St. Louis, MO, USA). The solution was passed through a 70 μm filter (Corning, Corning, NY, USA) to remove undissociated tissue and neutralized by the addition of a complete medium. The stromal cell pellet was obtained by centrifugation for 5 min and resuspended in the complete medium. After 24 h, the nonadherent cells were eliminated by changing the medium. Cultures were maintained at subconfluent levels in a 37°C incubator with 5% CO₂ and passaged 2-4 times with trypsin/EDTA (Nacalai Tesque, Kyoto, Japan) before use.

ASCs were isolated from the subcutaneous fat from patients consented in accordance with the Ethics Committee at the Tongji Medical College of Huazhong University of Science and Technology. The fresh fat specimen was washed three times with PBS containing 1% penicillin/streptomycin and chopped by sterile operation scissors. The chopped tissue was digested with 0.2% collagenase type I (Sigma, USA) at 37 °C for 1.5 h and centrifuged at 1500 rpm for 10 min. The cell pellet was resuspended and filtered through a 70-μm filter (Corning, USA). After another centrifugation for 5 min, the cell pellet was resuspended in the culture medium consisting of Dulbecco’s modified Eagle’s medium (DMEM, Gibco, USA) high glucose, 10% fetal bovine serum (FBS, Serapro, USA), and 1% penicillin/streptomycin. The culture medium was replaced 48 h after seeding to remove nonadherent cells; thereafter, the medium was replenished every 2–3 days. The ASCs at passages 2–7 were used for the following experiments. HaCAT (#GDC106, CCTCC) and human umbilical vein endothelial cells (HUVECs) (#GDC166, CCTCC) were obtained from the China Center for Type Culture Collection (CCTCC, Wuhan, China) and cultured following the manufacturer’s instructions. Human foreskin fibroblasts were isolated using previously described protocols [19 (link)].

Pancreata from 8- to 12-week-old female mice were perfused with 1000 U/ml Collagenase XI (Sigma-Aldrich, Oakville, ON, Canada) and incubated for 15 min at 37 °C. Pancreata were manually disrupted and passed through a 70 μM filter (Corning, New York, NY, USA) and islets were hand-picked. Islets were cultured in RPMI 1640 (11 mM glucose) supplemented with 10% FBS, 50U/ml penicillin/streptomycin and 2 mM l-glutamine at 37° in a 5% CO₂ humidified incubator. All tissue culture reagents were purchased from ThermoFisher (Burlington, ON, Canada). Islets were transduced with the shScramble or shMyt3 adenoviruses as done previously^{12 (link)} and cultured for 48 h prior to transplantation.