Erythrocyte lysate

The isolation, differentiation, and Oil Red O staining of hADSCs or mADSCs were conducted as described in our previous study^{34 (link)}. Briefly, 12 g of fresh abdominal SATs were isolated from four normal BMI (18.5–23.9) patients and iWAT was isolated from C57BL/6 male mice (n = 4). Adipose tissues were washed in phosphate buffer saline (PBS) three times and the tissue was subsequently cut into 1 mm × 1 mm size, followed by incubation in collagenase I solution (Gibco, Life Technology, China) for 1.5 h at 37 °C. The digested tissue mixture was filtered using a 70 µm cell strainer (BD Falcon, Becton Dickinson, Franklin Lakes, NJ, USA), centrifuged at 150 × g for 10 min and then the supernatant was poured off. Erythrocyte lysate (3 ml; Beyotime Institute of Biotechnology, Shanghai, China) was added into the sample for 3 min at room temperature, centrifuged at 150 × g for 10 min again, and the precipitate was washed by PBS thrice. Finally, the sample was cultured in Dulbecco’s modified Eagle medium/F12 (Life Technologies, Carlsbad, CA, USA) and supplemented with 10% fetal bovine serum (Life Technologies). The cellular lipid droplets content was extracted by 100% isopropanol and absorbance quantification was detected by using a spectrometer at 520 nM (Multiskan FC; Thermo Fisher Scientific, Inc., Waltham, MA, USA).

Porcine ovaries were collected from a local slaughterhouse under veterinarian control. All experimental procedures were performed with the approval of the Ethical Committee of Animal Experiments, Hunan Agricultural University (No. 43201701). Ovaries were kept in sterile saline containing 2% penicillin-streptomycin (Beyotime Biotechnology, Shanghai, China) at 37 °C and transported to the laboratory within 1 h. Ovarian granulosa cells were isolated and cultured according to the techniques described by others [19 (link)] with minor modifications. Briefly, follicular fluid was collected from 3-mm to 5-mm diameter follicles. Cells precipitated naturally in 37 °C water bath for 15 min followed by centrifuge. Cell pellet was resuspended in erythrocyte lysate (Beyotime, Shanghai, China) to remove blood cells. Then, cells were washed with PBS twice, collected, and cultured in DMEM medium supplemented with 10% fetal bovine serum (FBS) (Gibco-Invitrogen, Carlsbad, CA, USA) and 1% penicillin-streptomycin at 37 °C and 5% CO₂ in a humidified incubator (Thermo Fisher Scientific, MA, USA). The isolated cells were subjected to screening for animal pathogens including PRV, African swine fever virus (ASFV), and porcine circovirus type 2 (PCV2). Cells were also identified using a specific marker follicle-stimulating hormone receptor (FSH-R) by indirect immunofluorescence assay (IFA).