Dme f12

Lung tissue was enzymatically dissociated by incubation in 2mg/ml collagenase (Sigma, Poole, UK) in serum free phenol red free Dulbecco's Modified Eagle Medium: Nutrient Mixture F-12 (DME-F12, Life Technologies Ltd, Paisley, UK) medium for 2–24 hours at 37°C, with occasional agitation. Cells released were washed in DME-F12 medium containing 10% foetal bovine serum, plated in the same medium and cultured on tissue culture treated plastic at 37°C and 5% CO₂. Adherent cells were seen after 24 hours. Cells were used between passages 1 and 3. 621-101 cells are a TSC2-null cell line derived from a renal angiomyolipoma of a LAM patient and were a gift from Elizabeth Henske [19 (link)]. These cells were maintained in DME-F12 with 10% FCS. Normal Human Lung Fibroblasts (NHLFs) were purchased from Lonza (Slough, UK) and were maintained in DME-F12 with 10% FCS.

Briefly, the inguinal fat pad was removed from the abdominal cavity of rats anesthetized with diethyl ether. The adipose tissue was digested at 37 °C for 70 min using type I collagenase (Sigma, USA). After centrifugation at 170 × g for 10 min, the cells were resuspended in the lower layer using DME/F12 (HyClone, USA), and then filtered through a 70 μm mesh. The collected cells were cultured in a 37 °C, 5% CO₂ incubator in DME/F12 containing 10% FBS (Gibco, USA). After 48 h, the medium was changed for the first time. When the cells reached confluence, the adherent cells were detached with trypsin/ethylenediaminetetraacetic acid (EDTA) (Sigma, USA) and reseeded for expansion^{13 (link)}. When the ADSCs reached the third generation, Res at a concentration of 20 μM was added and cells were preincubated for 24 h for subsequent experiments.

BMSCs from the eGFP mice were extracted using the whole bone marrow apposition method. The eGFP mice were anesthetized intraperitoneally with 3% pentobarbital (30 mg/kg). Bilateral femurs and tibias were dissected after removing surrounding tissues and muscles, and rinsed with phosphate- buffered saline (PBS, Solarbio, China) for three times. Both ends of the bone were cut using tissue scissors, and following procedures were successively performed: (i) the bone marrow cavity was flushed three times using a 1 mL syringe; (ii) the culture medium containing bone marrow was collected; (iii) he supernatant was discard after centrifugation; (iv) the cell pellet was resuspended in DME/F-12 (Gibco, US); and (v) the resuspended cells were used to inoculate T25 culture flasks (Corning, US) to form a single cell layer. The cells were incubated at 37°C in a 5% CO₂ cell incubator and then the medium was replaced with fresh medium 4 h later, and every three days thereafter. Cells would be passaged when they reached a confluency of 80%, and the 3^rd to 5^th passaged cells(P3-P5) were used in subsequent experiments. BMSCs were inoculated on six-well plates (Corning, US) and the expression of their important markers CD90, CD34, and CD44 (BOSTER, China) were identified by immunofluorescence techniques.