Trypsin pbs

The isolation and culture of mOSE cells was done as previously described^{10 (link)}, in accordance with the guidelines of the Canadian Council on Animal Care and under a protocol approved by the University of Ottawa Animal Care Committee. Briefly, ovaries from randomly cycling female mice (FVB/N, 6 weeks old) were collected and incubated in 0.25% Trypsin/PBS (Invitrogen) (37 °C, 5% CO₂, 30 min) to facilitate OSE removal. mOSE cells were isolated by centrifugation and plated onto tissue culture plates (Corning) in mOSE media [a-Minimum Essential Medium (Corning) supplemented with 4% FBS, 0.01 mg/mL insulin-transferrin-sodium-selenite solution (ITSS; Roche), and 2 µg/mL EGF (R&D Systems)]. hOSE cells were isolated and cultured as previously described^{37 (link)}, with patient consent and under a protocol approved by the Ottawa Health Science Network Research Ethics Board (Protocol #1999540). Briefly, ovaries from 5 different women were collected during surgery for reasons other than ovarian pathology. Using a scalpel, hOSE cells were scraped from the ovarian surface and isolated by centrifugation in hOSE media (Wisent Bioproducts) supplemented with 10% FBS. All mouse and human OSE cells were passaged 2–3 times prior to experimental use and experiments were conducted with cells of a passage number less than 25.

Primary free-floating mOSE spheres were collected and washed in PBS. Spheres were dissociated by first incubating in trypsin/PBS (Invitrogen) at 37 °C for 10 min, then by passing cells through a 25 gauge needle to obtain a single cell suspension. Single cell suspension was verified using phase contrast microscopy. Cells were washed in PBS, counted using a hemocytometer, and plated in stem cell media at 5 × 10⁴ cells/mL. Cells were incubated in non-adherent 24-well culture plates (Corning) at 37 °C, 5% CO₂ for 14 days. Spheres were quantified using ImageJ using a pixel cutoff of >500 pixels and a circularity limit of 0.5–1.0.

To further study the mechanism of LMHFV and HMB on the fat metabolism in vitro, MDSCs were isolated from muscle tissues. MDSCs were isolated following Gharaibeh et al.'s protocol.25 Gastrocnemii of SAMP8 mouse were isolated, minced, and washed with Hank's Balanced Salt Solution (Invitrogen). It was re‐suspended in 10 mL of 0.2% collagenase‐type XI (Sigma) and incubated in a 5% CO₂ incubator for 1 h at 37°C. The mixture was then centrifuged, and supernatant was removed and incubated in 10 mL of dispose solution in 5% CO₂ incubator at 37°C for 45 min. The mixture was centrifuged again, supernatant removed and incubated in 10 mL 0.05% Trypsin‐PBS (Invitrogen) for 30 min at 37°C, centrifuged again and re‐suspended in proliferation media [DMEM, 10% FBS, 10% (vol/vol) horse serum (Invitrogen), 0.5% (vol/vol) chick embryo extract (USbio, USA)]. The cell resuspension was passed through a cell strainer (BD Biosciences, CA, USA) and the passed cells were put in a collagen‐coated T‐25 flask (Corning, NY, USA) marked as PP1 and incubated in 5% CO₂ incubator at 37°C for 2 h. The floating cells in the supernatant were transferred to another flask and incubated in 5% CO₂ incubator at 37°C for 24 h, marked as PP2. The manipulation was repeated every 24 h until PP6 was created. The cells were viewed as slow adhering cells that contained MDSCs for further application.