Bovine serum albumin bsa

The epidydymal fat tissue was isolated from euthanized mice, rinsed in PBS, weighed, and minced into small pieces (∼1 mm). The minced tissue was digested in KRH buffer (4.8 mM KCl, 2.5 mM CaCl₂, 1.2 mM MgSO₄, 118 mM NaCl, 20 mM HEPES [pH 7.5]) supplemented with 1.5% bovine serum albumin (BSA) (SeraCare Life Sciences), 30 µg/ml Liberase (Roche Applied Science, Indianapolis, IN), and 1 µg/ml DNaseI (Sigma-Aldrich) at 37°C for 25∼40 minutes with vigorous shaking. The digested samples were strained through a sterile 250 µm nylon mesh (Sefar, Buffalo, NY) and centrifuged at 200 g for 10 min. The floating cells were collected as the adipocyte fraction and the pelleted cells were collected as the stromal vascular fraction (SVF). The adipocytes were washed twice with KRH buffer +1.5% BSA before being processed for RNA extraction. The SVF was resuspended in ACK lysis buffer (150 mM NH₄Cl, 10 mM KHCO₃, 0.1 mM EDTA) and incubated at room temperature for 2 minutes to deplete erythrocytes, washed twice with KRH buffer +1.5% BSA, and finally resuspended in FACS buffer (1% BSA, 2 mM EDTA in 1×PBS) for flow cytometry or processed for RNA extraction.

TGF-β2, EGF and FGF21 groups were supplemented with recombinant human TGF-β2 (97.3% homology with rat), recombinant rat EGF and recombinant human FGF21 (92% homology with rat) (all from Peprotech, Rocky Hill, NJ, USA). These products were reconstituted in phosphate-buffered solution (PBS, pH 7.2) with 0.1% bovine serum albumin (BSA, SeraCare Life Sciences, Milford, MA, USA), according to the manufacturer’s recommendations. The dose of TGF-β2 was 35 μg/kg/day, which was based on the amount of TGF-β2 found in mid-lactation rat milk and milk intake by pups at 4–14 days of age [13 (link)]. The dose of EGF was 100 μg/kg/day, which was demonstrated to be effective as a treatment in a rat model of NEC [29 (link)]. Finally, the dose of FGF21 was 5 μg/kg/day, an amount that was established in relation to TGF-β2, which was found in a 1:10 ratio FGF21:TGF-β2 [23 (link),30 (link)]. The REF group received a matched volume of the vehicle (1% BSA in PBS) used to dilute the GF.