Dulbecco s modi ed eagle s medium dmem

Our lab washed adipose tissue harvested from healthy people or normal mice with phosphate-buffered saline (PBS) and minced it before digestion with 0.2% collagenase I (Sigma-Aldrich, St. Louis, MO, USA) for 1 h at 37°C with intermittent shaking. Technician washed tissue that digested with Dulbecco's Modi ed Eagle's Medium (DMEM; Sigma-Aldrich) including fetal bovine serum (FBS, Gibco BRL, Frederick, MD, USA) of 15%, and centrifuged it at 1000 rpm for 10 min to eliminate mature adipocytes. Our lab resuspended pellet in DMEM supplied with FBS of 15%, 100 U/mL penicillin, and 100 μg/mL streptomycin and cultured it at 37°C with CO 2 of 5%. When the ADSCs had reached 80%~90% con uency, our lab detached them with 0.02% ethylenediaminetetraacetic acid (EDTA)/0.25% trypsin (Sigma-Aldrich) for 5 min at room temperature and then replated them. For phenotypic analyses, we leveraged uorescein isothiocyanate (FITC)-conjugated CD29, CD44, CD90, CD105, and vWF antibodies. An IgG-matched isotype was employed as internal control for every antibody. Normoxic ADSC cultures were in 95% air (20% O 2 ) and CO 2 of 5%. For hypoxia induction, our lab cultured ADSCs in 94% N 2 , 1% O 2 , and CO 2 of 5%.

Our lab washed adipose tissue harvested from healthy people or normal mice with phosphate-buffered saline (PBS) and minced it before digestion with 0.2% collagenase I (Sigma-Aldrich, St. Louis, MO, USA) for 1 h at 37°C with intermittent shaking. Technician washed tissue that digested with Dulbecco's Modi ed Eagle's Medium (DMEM; Sigma-Aldrich) including fetal bovine serum (FBS, Gibco BRL, Frederick, MD, USA) of 15%, and centrifuged it at 1000 rpm for 10 min to eliminate mature adipocytes. Our lab resuspended pellet in DMEM supplied with FBS of 15%, 100 U/mL penicillin, and 100 μg/mL streptomycin and cultured it at 37°C with CO 2 of 5%. When the ADSCs had reached 80%~90% con uency, our lab detached them with 0.02% ethylenediaminetetraacetic acid (EDTA)/0.25% trypsin (Sigma-Aldrich) for 5 min at room temperature and then replated them. For phenotypic analyses, we leveraged uorescein isothiocyanate (FITC)-conjugated CD29, CD44, CD90, CD105, and vWF antibodies. An IgG-matched isotype was employed as internal control for every antibody. Normoxic ADSC cultures were in 95% air (20% O 2 ) and CO 2 of 5%. For hypoxia induction, our lab cultured ADSCs in 94% N 2 , 1% O 2 , and CO 2 of 5%.

Human pancreatic cancer cells PANC1, Pa-Tu-8988, BXPC-3, HEK293T, CFPAC, and Mia-CaPa2 were purchased from the Cell Bank of the Chinese Academy of Science (Shanghai, China) and human pancreatic ductal epithelial cells hTERT-HPNE cell line was obtained from BaiRong Biotechnology (Shanghai, China). All the cell lines, which were authenticated via short tandem repeat pro ling analysis using Genetic Testing Biotechnology (Suzhou, China), were cultured in Dulbecco's modi ed Eagle's medium (DMEM) (Sigma-Aldrich, St. Louis, MO, USA) supplemented with 10% fetal bovine serum (FBS) (Sigma-Aldrich), 100 U/ml penicillin (Beyotime, Shanghai, China), 0.1 mg/ml streptomycin (Beyotime), and amphotericin B (Beyotime). Further, all the cell lines were incubated at 37°C in a 5% CO 2 atmosphere.

We obtained mouse embryonic broblasts (MEFs) from ATCC (cat. no. SCRC-1040), NIH3T3 broblasts from ATCC (cat. no. CRL-1658) and HAP1 chronic myeloid leukemia cells from Horizon Discovery (cat. no. C859). We grew MEFs in Dulbecco's Modi ed Eagle's Medium (DMEM) (Sigma, cat. no. D6429) supplemented with 15% fetal bovine serum (FBS) (Sigma, cat. no. F9665), NIH3T3 cells in DMEM supplemented with 10% FBS, and HAP1 cells in Iscove's Modi ed Dulbecco's Medium (Sigma, cat. no. I6529) supplemented with 10% FBS. None of these cell lines is included in the ICLAC database of commonly misidenti ed cell lines. We regularly checked the cells for Mycoplasma contamination but did not authenticate them. We incubated the cells at 37°C in 5% O 2 and 5% CO 2 . We grew the cells until they reached 80% con uency on coverslips (VWR, cat. no. 630-2185) in the case of MEFs and on 9-well chambered coverslips (custom-made by Grace Bio-Labs) in the case of HAP1 cells. We then processed the cells following an adapted version of the 3D-FISH protocol 30 , as described in Supplementary Methods.