Adipose tissue progenitor isolation kit

Bone marrow cells were suspended with PBS containing 0.5% Bovine Serum Albumin (BSA) and 2 mM EDTA. Then an Adipose Tissue Progenitor Isolation kit (Miltenyi Biotec, San Diego, CA, USA) was used to isolate the adipocyte progenitor cells according to the manufacturer’s instruction. Briefly, cell suspension was incubated with Depletion Cocktail at 4 °C in the dark for 15 min. Then total reaction volume was adjusted to 500 μL, and cell suspension was applied onto a pretreated LS Column placed in the MACS Separator. Flow-through cells were collected and incubated with Isolation Cocktail at 4 °C in the dark for 15 min. Further positive selection was performed using a MS Column. After being fully washed, the positive cells were flushed out from the MS Column.

Epididymal, inguinal, and axillary fat pads were dissected immediately after mice were sacrificed. The fat pads were incubated in Hank’s Balanced Salt Solution (Gibco) containing 3% (w/v) bovine serum albumin (Gold Biotechnology; St. Louis, MO, USA) for 15 min at room temperature, followed by centrifugation at 200× g for 7 min. The fat pads were digested with 0.1% (w/v) collagenase II (Gibco; Waltham, MA, USA) in 37 °C for 60 min followed by filtering through 70 µm cell strainer. Adipose progenitor cells (APCs) were then enriched using Adipose Tissue Progenitor Isolation Kit (Miltenyi Biotec; Bergisch Gladbach, Germany) according to the manufacturer’s protocol.

Figs. 1 and S2: Male C57BL/6N background mice were injected at ZT0 or ZT12 (n = 4 per ZT) with EdU as described above and then euthanized at ZT4 or ZT16, respectively. Following euthanasia, the SVF from iWAT and eWAT were isolated as described in the “White adipose tissue fractionation” section. The SVF pellet was washed and fractionated into adipose progenitor cells (APC⁺) or non-adipose progenitor cells (APC⁻) with the adipose tissue progenitor isolation kit (Miltenyi Biotec 5200501358) following manufacturer directions. Cell fractions then were fixed and labeled with Click-iT EdU Alexa Fluor 647 kit (Invitrogen C10424) and subsequently analyzed by cell cytometry. A separate cohort of age-matched mice were sacrificed at ZT0 or ZT12 and both APC⁺ and APC⁻ fractions were isolated from the iWAT and eWAT and immediately frozen at −80 °C for further gene expression analysis.