HUVECs and GFP-HUVECs were used between passages 4 to 7. HUVECs, GFP-HUVECs, and HMEC-1 cells were grown in complete MCDB131® medium (Life Technologies, Rockville, MD) supplemented with 20% fetal bovine serum (FBS), endothelial cell growth supplement, 2mmol/l glutamine, heparin, and gentamicin and incubated at 37°C and 5% CO2. Primary human lung fibroblasts were cultured in MEM medium (Life Technologies, Rockville, MD) supplemented with 10% FBS and antibiotics. HAVICs, generated as previously described (Yu et al., 2017 (link)), were used at passages 3–5 and cultured in DMEM medium supplemented with 10% FBS, glutamine, and antibiotics. HASMCs were cultured in smooth muscle cell growth medium (SmGM™ 2; Lonza, Basel, Switzerland) supplemented with 5% FBS, insulin, FGF-B, gentamycin, amphotericin, and epithelial growth factor (EGF).
Complete mcdb131 medium
Manufactured by Thermo Fisher Scientific
Sourced in United States
MCDB131 medium is a complete, serum-free, and chemically defined cell culture medium designed to support the growth and maintenance of a variety of cell types, including those derived from human or animal sources. The medium provides essential nutrients, vitamins, and growth factors required for optimal cell performance in vitro.
Automatically generated - may contain errors
Lab products found in correlation
Mem medium, by Thermo Fisher Scientific (1 mentions)
Smgm 2, by Lonza (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Glutamax, by Thermo Fisher Scientific (1 mentions)
Complete high glucose dmem, by Thermo Fisher Scientific (1 mentions)
Hmecs, by Thermo Fisher Scientific (1 mentions)
Hmecs, by American Type Culture Collection (1 mentions)
5 protocols using complete mcdb131 medium
1
Culturing Endothelial and Fibroblast Cells
2
HUVEC Cell Culture Conditions
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HUVECs were maintained in complete MCDB 131 medium (Life Technologies, Rockville, MD, USA) supplemented with 20% fetal bovine serum (FBS), EC growth supplement, 2 mM glutamine, heparin, and gentamicin sulfate, and incubated at 37 °C and 5% CO2. In experiments where EC migration, survival, and activation of caspase-3 activity were measured, cells were cultured in basal MCDB131 medium containing gentamicin sulfate and 2% FBS.
3
Raman spectroscopy of CQ-treated HMEC-1 cells
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Human dermal microvascular endothelial cells (HMEC-1) were maintained in complete MCDB131 medium (Gibco, Life Technologies, Grand Island, NY, USA) at 37 °C/5% CO2. HMEC-1 (3rd passage) cells were seeded at a concentration of 18 × 104 and left for 24 h on CaF2 slides (Crystran LTD, Poole, Dorset, UK). For Raman measurements, cells were treated with various concentrations of CQ for 24 h. Then cells were fixed using 2.5% solution of glutaraldehyde in phosphate-buffered saline (PBS; Gibco, Life Technologies, Grand Island, NY, USA) for 5 min. The cell density seeded for fluorescence imaging was 3 × 104 per well.
4
Fasting and Refeeding Effects on Angiogenesis and Proliferation
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HMECs were normally grown in complete MCDB131 medium (Gibco, Grand Island, USA) containing 10% FBS (Gibco) and 1% GlutaMAX (Gibco). JB6 and NIH3T3 were normally cultured in complete high-glucose DMEM (Gibco) containing 10% FBS (Gibco). To assess the effects of fasting and refeeding on the angiogenic activities of HMECs and proliferation of JB6 and NIH3T3, these cells were divided into four groups: 1) N24 group: cells were cultured in complete MCDB131 or DMEM media for 24 h; 2) F24 group: cells were cultured in serum-free MCDB131 or DMEM media for 24 h; 3) F24N24 group: cells were cultured in serum-free MCDB131 or DMEM media for 24 h and then incubated in serum-containing complete MCDB131 or DMEM media for another 24 h; 4) N48 group: cell were cultured in complete MCDB131 or DMEM media for 48 h. Cultures were maintained at 37 °C in 5% CO2.
5
LIPUS Enhances Endothelial Cell Function
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Human microvascular endothelial cells (HMECs) were purchased from the American Type Culture Collection (ATCC, Manassas, USA) and used for in vitro experiments. The HMECs were reseeded into six-well cell culture plates overnight in complete MCDB 131 medium (Gibco, Waltham, USA) and then stimulated with LIPUS (Fig. 1D). The in vitro-cultured cells were in a fragile external environment different from the ones in vivo, and a previous study showed that an intensity over 200 mW/cm 2 might be harmful to cultured endothelial cells. 17 (link) Thus, the parameters for cell treatment were set as follows: frequency of 1 MHz, intensity of 100 mW/cm 2 and duration of 10 min per day for 3 days. Control cells were subjected to the same treatment with the machine turned off. After the last stimulation, the cells were cultured in complete medium for another 30 min and harvested for tube formation assays and protein extraction.
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