Collagenase nb4g

Animals were anesthetized with ketamine/xylazine (100/12.5 mg/kg). Subcutaneous adipose tissue from the inguinal region was obtained and weighted in sterile conditions. The tissue was washed with sterile phosphate-buffered saline (PBS) and was enzymatically processed by collagenase (Collagenase NB 4G, Serva, Iceland, NY, USA) solution (0.9 units/ml) incubated at 37°C for 150 min on an orbital shaker. Then, an equal volume of “stop medium” (Dulbecco's modified Eagle's medium (DMEM)) (Sigma-Aldrich, Saint Louis, MO, USA) with 20% fetal bovine serum (FBS) (Sigma-Aldrich) was added; then, samples were centrifuged at 600 × g for 10 min at 4°C. The pellet was resuspended in culture media (DMEM+10% FBS+1% penicillin-streptomycin), and the cell suspension was filtered through a 40 μm pore-size filter to obtain the stromal vascular fraction (SVF).

Epididymal fat pads were harvested from GFP⁺ donor mice, transferred into 10% Dulbecco’s modified eagle medium (DMEM; 100 U/mL penicillin, 0.1 mg/mL streptomycin; Biochrom GmbH, Berlin, Germany), and washed with phosphate-buffered saline (PBS; Biochrom GmbH). The fat was mechanically minced and digested for 10 min with collagenase NB4G (0.5 U/mL; Serva Electrophoresis GmbH, Heidelberg, Germany) while stirring under humidified atmospheric conditions (37 °C, 5% CO₂). The digestion was neutralized with PBS supplemented with 20% fetal calf serum (FCS; Biochrom GmbH) and the cell-vessel suspension was incubated for 5 min at 37 °C. After removal of fat supernatant, the remaining cell-vessel suspension was filtered through a 500 μm mesh (pluriStrainer; pluriSelect Life Science, Leipzig, Germany) and centrifuged for 5 min at 600 × g to obtain a pellet.

These experiments were carried out in accordance with the protocol approved by the Committee on Animal Research at Nara Institute of Science and Technology and the RIKEN Animal Experiment Committee. Subcutaneous fat tissues from three- to four-month-old ICR male mice (n = 3 per sample, two biological replicate samples) were minced into small pieces and incubated with 0.4 U/mL collagenase NB4G (Serva) at 37 °C for 35 min in a shaking water bath. The digested solution was sequentially filtered through 100- and 40-μm cell strainers (Corning), followed by centrifugation at 250×g for 5 min to remove mature adipocytes. The pellet was treated with erythrocyte lysis buffer (BD Biosciences) and centrifuged at 180×g for 5 min. The nucleated cells were suspended in HBSS with 0.1% BSA, filtered through a 20-μm cell strainer (pluriSelect), and then kept on ice (Cell solution A). The cell aggregates that did not pass through the 20-μm strainer were further treated with Accutase (Thermo Fisher Scientific) at 37 °C for 15 min to dissociate them into single cells, centrifuged at 180×g for 5 min, and suspended in HBSS with 0.1% BSA (Cell solution B). Cell solutions A and B were mixed and again filtered through the 20-μm cell strainer, followed by centrifugation at 180×g for 5 min. The pellet was resuspended with HBSS with 0.1% BSA, stained with PI, and used for single-cell analysis.

Lipoaspirates were washed with PBS and enzymatically processed by collagenase (Collagenase NB 4G, Serva, Iceland, NY, USA) solution incubated (0.3 units/ml) at 37°C for 120 min in an orbital shaker. Then, an equal volume of “stop medium” (DMEM) with 20% FBS was added and samples were centrifuged at 600 × g for 10 min at 4°C. The pellet was resuspended in DMEM+10% FBS+1% penicillin-streptomycin, and the cell suspension was filtered through a 100 μm pore-size filter. This last step was repeated but using a 40 μm pore-size filter to obtain human SVF.