100 mm cell strainer

Waste LPI fractions were received from the SNBTS islet isolation lab. LPIs were washed once in SMPL medium, centrifuged at 300 £ g for 5 min and then cultured at 0.006 mL/cm 2 at 37°C in 5% CO 2 in SMPL medium (e.g., 0.45 mL LPI fraction in a T-75 flask plus 9.5 mL SMPL medium). Explant outgrowth was assessed, and adherent cells were observed migrating from the explanted materials. The medium was carefully exchanged at day 7 and thereafter changed every 3À4 days. Cultures were observed and photographed using an EVOS cell imaging system (Thermo Fisher Scientific). Once the cultures had reached 80À90% confluence, the cells were recovered with a 10-min incubation at 37°C with 0.13 mL/cm 2 TrypLE Select £1 (Thermo Fisher Scientific). To remove cell debris, the material was passed through a 100-mm cell strainer (Falcon). The cells were counted using a hemocytometer and designated passage 0. These cells were either cryopreserved at 1 £ 10 6 per 2-mL cryovial in CryoStor CS10 (Sigma-Aldrich) or re-cultured at a density of 3000 cells/cm 2 in CellBIND flasks (Corning).

Embryo heads at embryonic day (E) 9.5 were dissected from the body below the first branchial arch and collected in DMEM containing 10% FBS. Since Wnt1-Cre is expressed in the dorsal neural tube, the neural tube was removed from embryos obtained from this cross (Supplementary Fig. S1A). Two to four GFP positive heads were pooled for each sample and digested with 100 μL Trypsin (0.025%, Gibco Life Technologies), 20 μL collagenase (1 mg/mL, Company) and 80 μL PBS (Life Technologies) for 5-10 min at 37 °C and 5% CO 2 and then mechanically dissociated by pipetting. Following a second 10 min incubation the tissues were again mechanically dissociated. Prior to cell sorting, 500 μL PBS/ EDTA (1:1 500 mM EDTA: PBS) was added to the dissociated cells and they were filtered through a 100 mm cell strainer (Falcon). Cell sorting yielded 4000-18000 GFP-positive cells per sample, which were centrifuged at 4000 rpm for 5 min at 4 °C and the pellet washed with cold PBS, followed by further centrifugation at 4000 rpm for 5 min at 4 °C. The cell pellet was snap frozen in liquid nitrogen and stored at À 80 °C.

Epididymal fat pads were removed, minced, and digested using collagenase type II (Calbiochem) at 37 C for 1 hr in Krebs-Ringer-HEPES-bicarbonate (KRH) buffer supplemented with BSA (3.5%; Roche). The digested tissue was filtered through a 100 mm cell strainer (BD Falcon), and the floating adipocytes were recovered and used for RNA or protein extraction.

Isolation and culture of human adipose tissue-derived mesenchymal stem cells (hASCs) hASCs were collected from three independent female donors as waste material during tumescent abdominal liposuction as described in detail previously. (21, 39, 40) All patients had given prior written consent. For isolation of hASCs, the harvested lipoaspirate was first washed with PBS (PAA, Dartmouth, MA, USA) to remove cell debris, then digested with collagenase NB4 (Serva, Heidelberg, Germany) for 1 hour to further disintegrate adipose tissue agglomerates. Subsequent centrifugation resulted in a cell pellet that contained the hASCs. The cell pellet was resuspended in erythrocyte lysis buffer to eliminate red blood cells, and washed with PBS. Cells were next filtered through a 100-mm cell strainer (BD, Vienna, Austria) and cultured at 37°C, 5% CO 2 and 95% air humid in DMEM-low glucose/HAM's F-12 medium (GE Healthcare, Vienna, Austria) supplemented with 10% fetal calf serum (FCS; Sigma-Aldrich, Munich, Germany), 4 mM L-glutamine, and 1 ng/ mL recombinant human basic fibroblast growth factor (rhFGF; R&D Systems, Minneapolis, MN, USA) as published. (21) Medium was refreshed twice a week and cells were utilized for further experiments or cryopreservation on reaching 3 Â 10 5 cells/plate. All the cells used in the performed experiments were within one to three passages.