α minimum essential media α mem

Four- to 6-week-old ICR mice were used in the study. Animal care and experiments were conducted under the approval of the Animal Care Committees of Zhejiang University. Bone marrow was isolated from the femurs. Red blood cells were lysed with 0.8 % ammonium chloride solution, and then the cells were washed, collected for 3D differentiation systems. OP9 (ATCC) and OP9DL1 (a gift from Professor Cheng Tao, State Key Lab of Experimental Hematology Institute of Hematology and Hospital of Blood Diseases, Tianjin, China) were expanded with α-minimum essential media (α-MEM) (Gibco) supplemented with 20 % fetal bovine serum (FBS; Hyclone) at 37 °C in 5 % CO₂ atmosphere. The stromal cells were inactivated by mitomycin C treatment for 2 h (10 mg/ml) and prepared for 3D induction systems.

Adipose tissue biopsies of the abdominal subcutaneous region were performed under local anesthesia [35 (link)]. Adipose biopsies were taken in the overnight fasted state between 8 and 10 AM. The scWAT was digested with collagenase (type 1, 1 mg/2ml in HBSS) for 30-60 min at 37° C with shaking (100rpm), as described previously [36 (link)]. The mixture was passed through a 250-micron mesh, centrifuged at 500g for five minutes and floating adipocytes were discarded. Cell pellets were treated with erythrocyte lysis buffer (BioLegend, Cat: 420301) for five to 10 minutes at room temperature followed by centrifugation at 500g for five minutes. After discarding the supernatant, cells were resuspended in proliferation media (PM) (Gibco α-Minimum Essential Media (αMEM), 10% Fetal Bovine Serum (FBS) and 1% Penicillin-Streptomycin), then plated and cultured in an incubator at 37° C at 5% carbon dioxide. Media was replaced after four hours to remove blood cells. APCs continue to grow with PM changes every other day until they reached ~80% confluency, then they were frozen in freezing media (10% FBS, 10% DMSO in αMEM) and stored in dewars containing liquid nitrogen.

We purchased human dental pulp stem cells (hDPSCs) collected from human sound third molars (Lonza, Basel, Switzerland). These cells have been reported to contain mesenchymal-like cells. Surface antigens on these cells have been confirmed for cluster designation (CD) 29+, CD73+, CD90+, CD105+, CD166+, CD34-, CD45-, and CD133- by flow cytometry. Cells were collected (TrypLE Select, Life Technologies, Carlsbad, CA, USA) when they reached 80% confluence. For the following assay, hDPSCs (passages 2–4) were cultured in α-Minimum Essential Media (α-MEM; Life Technologies, Carlsbad, CA, USA) supplemented with penicillin-streptomycin 10 µg/mL (Sigma-Aldrich, St. Louis, MO, USA). In the in vitro assays, 1% fetal bovine serum (FBS; Sigma-Aldrich, St. Louis, MO, USA) was used.