Normal Human Dermal Fibroblasts (NHDF) were purchased from Sigma (C-12302, Sigma). Cells were cultured in EMEM (ECB2071L, Euroclone), 10% FBS (ECS0180L, Euroclone), 1% NEAA (ECB3054D, Euroclone), 1% 200 mM glutamine (ECB3000D, Euroclone), 1% Penicillin/Streptomycin (ECB3001D, Euroclone). Human aortic smooth muscle cells (HSMC) were purchased from the American Tissue Culture Collection (PCS-100-012, ATCC, Manassas, USA). Cells were cultured in ATCC Vascular Cell Basal Medium (PCS-100-030, ATCC; 500 mL added with 500 μL ascorbic acid, 500 μL rh EGF, 500 μL rh insulin and rh FGF-b, 25 mL glutamine), 5% FBS (ATCC Vascular Smooth Muscle Growth Kit), and 5 mL Penicillin-Streptomycin 100X (Euroclone, Milan, Italy). Human umbilical vein endothelial cells (HUVEC) were purchased from Lonza (Bend, OR, USA) and routinely grown in Endothelial Growth Medium (EGM™-2) as indicated by the provider. The neuroblastoma cell line SH-SY5Y was purchased from Sigma (ECACC 94030304, Sigma) and cultured in EMEM (ECB2071L, Euroclone) and HAM’S F-12 (ECB7502L, Euroclone) 1:1, 10% FBS (ECS0180L, Euroclone), 1% NEAA (ECB3054D, Euroclone), 1% 200 mM glutamine (ECB3000D, Euroclone), 1% Penicillin/Streptomycin (ECB3001D, Euroclone). All cultures were maintained at 37°C in a 5% CO2 incubator.
Ecb3000d
Manufactured by Euroclone
Sourced in Italy
The ECB3000D is a laboratory equipment designed for incubation and cultivation of cell cultures. It features a temperature-controlled environment for maintaining optimal conditions for cell growth and development.
Automatically generated - may contain errors
Lab products found in correlation
Ecb3001d, by Euroclone (12 mentions)
Ecs0180l, by Euroclone (9 mentions)
Ecb7501l, by Euroclone (4 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (4 mentions)
G8644, by Merck Group (3 mentions)
Ecb3001d, by Merck Group (3 mentions)
U3003, by Merck Group (3 mentions)
Ecb2071l, by Euroclone (2 mentions)
Ecm0542d, by Euroclone (2 mentions)
S8022, by Selleck Chemicals (2 mentions)
Bi 10773, by Selleck Chemicals (2 mentions)
Ecm0180d, by Euroclone (2 mentions)
Ecs5000l, by Euroclone (2 mentions)
Du145, by American Type Culture Collection (2 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (2 mentions)
B27 supplement, by Thermo Fisher Scientific (2 mentions)
Flp in 293 trex cells, by Thermo Fisher Scientific (1 mentions)
Dulbecco modified eagle medium (dmem), by Merck Group (1 mentions)
Penicillin, by Merck Group (1 mentions)
Streptomycin, by Merck Group (1 mentions)
18 protocols using ecb3000d
1
Cultivation of Common Cell Lines
2
Culturing and Characterizing Engineered HeLa and Flp-In 293 T-REx Cell Lines
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HeLa cells were cultured in MEM (catalogue no. ECB2071L, Euroclone) supplemented with 10% inactivated fetal bovine serum (FBS) (catalogue no. ECS0180L, Euroclone), 2 mM glutamine (catalogue no. ECB3000D, Euroclone), penicillin (100 IU ml−1) and streptomycin (100 μg ml−1) (catalogue no. ECB3001D, Euroclone) and maintained at 37 °C and 5% CO2. RagC KO HeLa cells and RagA KO HeLa cells were previously generated and described in ref. 7 (link). Flp-In 293 T-REx cells (catalogue no. R78007 Thermo Fisher) were grown in DMEM (catalogue no. D6429 Sigma-Aldrich), supplemented with 10% (vol/vol) FBS (catalogue no. 10270 Thermo Fisher), 100 U ml−1 penicillin and 100 µg ml−1 streptomycin (catalogue no. P0781 Sigma-Aldrich), 100 µg ml−1 Zeocin (catalogue no. ant-zn-5b InvivoGen, Toulouse, France) and 15 µg ml−1 Blasticidin (ant-bl-5b InvivoGen). Cell lines were validated by morphological analysis and routinely tested for absence of mycoplasma.
3
High Glucose-Induced Cardiomyocyte Dysfunction: Tirzepatide Evaluation
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AC16 human cardiomyocyte cell lines were purchased from EMD Millipore (cod. SCC109). Following the manufacturer's instructions, the cell line was tested and authenticated for mycoplasma contamination, resulting in negative data. Cells were cultured in Dulbecco's Modified Eagle's Medium /Nutrient Mixture F-12 (cod. D8437, Sigma) containing 12.5% fetal bovine serum (FBS) (cod. ECS0180L, Euroclone), 1% antibiotics penicillin–streptomycin (cod. ECB3001D, Euroclone), and 1% of L-glutamine (cod. ECB3000D, Euroclone). The cell line was maintained in the incubator at 37 °C and 5% CO2. The cells were grown between 4 and 6 passages, and experiments were performed in triplicate. AC16 were exposed to 33 mmol/L D glucose (cod. G8644, EMD Millipore) for 7 days [19 (link)] and treated with tirzepatide (LY3298176, selleckchem) at a concentration of 100 nM. The medium was changed every 48 h. Normal glucose, NG, was considered the cells exposed to normal glucose concentration (5.5 mmol/L) and cultured for 7 days. A dose–response curve, using cell viability and toxicity assay, was performed to evaluate the right concentration of tirzepatide to carry out experiments (Additional file 1 : figure).
4
Immortalized human mammary cell culture
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hTERT-immortalized human mammary epithelial cells (IMEC) and XD cells were cultured at 37 °C and 5% CO2 in 1:1 DMEM/F-12 medium (gibco #11320-074) supplemented with insulin (Clonetics, MEGM SingleQuots #CC-4136), EGF (Clonetics, MEGM SingleQuots #CC-4136), bovine pituitary extract (Clonetics, MEGM SingleQuots #CC-4136), hydrocortisone (Clonetics, MEGM SingleQuots #CC-4136) and 100 ng/ml cholera toxin (Sigma #8052). IMEC-MYC, IMEC-PIK3CAH1047R, IMEC-P53DD, and IMEC-RAS were generated by transducing IMEC with pMXs-c-Myc, PGK-PIK3CAH1047R, pBABE-puro-RAS V12, and MSCV-p53DD-iGFP vector, respectively. IMEC-MYC-7TGP cells were generated by transduction of IMEC-MYC with 7TGP vector. T47D and MCF7 cells were cultured at 37 °C and 5% CO2 in DMEM high glucose (Euroclone #ECB7501L) supplemented with 10% fetal bovine serum (Euroclone #ECS0180L), 1 mM sodium pyruvate (Euroclone #ECM0542D) and 2 mM glutamine (Euroclone #ECB3000D). ZR751 cells were cultured at 37 °C and 5% CO2 in RMPI Medium 1640 (gibco #31870-025) supplemented with 10% fetal bovine serum, 2 mM glutamine and 1 mM sodium pyruvate. MCF7-MYC, T47D-MYC, and ZR751-MYC were generated by transduction with pMXs-c-Myc.
5
IPEC-J2 Cell Culture Protocol
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IPEC-J2 cells (porcine jejunal epithelial cells, IZSLER Cell Bank code BS CL 205) were cultured in a complete medium consisting of a mixture (1:1) of Dulbecco’s modified Eagle high glucose medium (DMEM, cod. ECM0101L, Euroclone, Milan, Italy) and nutrient mixture F-12 (F12, cod. ECB7502L, Euroclone, Milan, Italy) enriched with 10% fetal bovine serum (FBS, GIBCO™, cod. A38401, Thermofisher scientific, Milan, Italy), 1% L-glutamine 2 mM solution (cod. ECB3000D, Euroclone, Milan, Italy) and 1% penicillin/streptomycin solution (cod. ECB3001D, Euroclone, Milan, Italy). Cells were seeded into 12 well plates (1 mL per well, 3 × 105 cells/mL; Euroclone, Milan, Italy) and then incubated at 37 °C, 5% CO2, until confluence.
6
Characterization of Cellular Bioenergetics
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All chemicals, including tosyl phenylalanyl chloromethyl ketone (TPCK)-treated trypsin, were from Thermo Scientific, unless otherwise stated. Acetonitrile (CH3CN, #15611400, Honeywell Riedel-de Haen), formic acid (FA, #10627431) and LC-MS grade water (#15651400, Honeywell Riedel-de Haen) were from Fisher Scientific. MC2494 was prepared as previously reported (17 (link)). Antibodies: PGC1α (#ab191838), PGC1β (#ab176328), and SOD2 (#ab13533) were from Abcam and GAPDH was from Santa Cruz (#sc-47724). Cell lines: U937 (#ACC5) human myeloid leukemia cells were purchased from DSMZ and MCF7 (#ICLCHTL95021) breast cancer cells from Cell Bank Interlab Cell Line Collection. U937 and MCF7 cells were propagated in RPMI (Euroclone #ECB9006) and DMEM (Euroclone #ECB7501), respectively, with 10% fetal bovine serum (FBS; Gibco #10270), 2 mM L-glutamine (Euroclone #ECB3000D), and antibiotics (100 U/mL penicillin, 100 μg/mL streptomycin, Euroclone #ECB3001D, and 250 ng/mL amphotericin-B; Euroclone #ECM0009). All cell lines were grown at 37°C with 5% CO2, and were then tested, authenticated, and used for 10–20 passages. Mycoplasma contamination was checked using EZ-PCR Mycoplasma Test Kit (Biological Industries #20-700-20).
7
High-Glucose-Induced Cardiomyocyte Dysfunction
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AC16 human cardiomyocyte cell lines were purchased from EMD Millipore (cod. SCC109). Following the manufacturer's instructions, the cell line was tested and authenticated for mycoplasma contamination, which was negative. Cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM)/F12 (cod. AL215A, Microgem) containing 12.5% fetal bovine serum (FBS) (cod. ECS0180L, Euroclone), 1% antibiotics penicillin-streptomycin (cod. ECB3001D, Euroclone), and 1% of L-glutamine (cod. ECB3000D, Euroclone). The cell line was maintained in the incubator at 37 °C and 5% CO2. The cells were grown between 5 and 7 passages, and experiments were performed in triplicate. AC16 were exposed to 33 mmol/L D glucose (cod. G8644, EMD Millipore) for 7 days and treated with EMPA at a concentration of 0.5 µM (cod. S8022, BI 10773, Selleckchem) [28 (link)]. The medium was changed every 48 h. Normal glucose (NG), considered the control, are cells exposed to normal glucose concentration (5.5 mmol/L) and cultured for 7 days.
8
Murine Endothelial and Osteoblastic Cell Culture
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Endothelial Cells (ECs) 1G11 [14 (link)], obtained from murine lung and Mouse Aortic Endothelial Cells GFP positive (MAEC GFP+), provided by Stefania Mitola (University of Brescia, Brescia, Italy), were cultured in endothelial growth medium composed of Dulbecco’s Modified Eagle’s Medium high glucose with sodium pyruvate (ECB7501L Euroclone, Milano, Italy), supplemented with 10% fetal bovine serum (ECS0180L Euroclone, Milano, Italy).
Osteoblastic cell line 2T3 [15 (link)], provided by Dr. Lynda Bonewald (Indiana University School of Medicine, Indianapolis, Indiana), was cultured in complete medium with a composition of Alpha Minimum Essential Medium (AU-L0476-500 Aurogene, Roma, Italy) supplemented with 10% Calf Serum (AU-S0400-500 Aurogene, Roma, Italy).
All media were supplemented withl -glutamine 0.02 mM (ECB3000D Euroclone, Milano, Italy) and 10 U·mL−1 penicillin/streptomicin (ECB3001D Euroclone, Milano, Italy).
Confluent cells (80–90%) were sub-cultured routinely at a split ratio of 1:8 three times a week, 1:5 three times a week, and 1:8 two times a week, respectively, for 1G11, MAEC GFP+, and 2T3, after trypsin/EDTA digestion and cultured at 37 °C with 5% CO2 in a humid atmosphere. Where indicated, cells were differentiated for a well-defined timing in an appropriate culture medium.
Osteoblastic cell line 2T3 [15 (link)], provided by Dr. Lynda Bonewald (Indiana University School of Medicine, Indianapolis, Indiana), was cultured in complete medium with a composition of Alpha Minimum Essential Medium (AU-L0476-500 Aurogene, Roma, Italy) supplemented with 10% Calf Serum (AU-S0400-500 Aurogene, Roma, Italy).
All media were supplemented with
Confluent cells (80–90%) were sub-cultured routinely at a split ratio of 1:8 three times a week, 1:5 three times a week, and 1:8 two times a week, respectively, for 1G11, MAEC GFP+, and 2T3, after trypsin/EDTA digestion and cultured at 37 °C with 5% CO2 in a humid atmosphere. Where indicated, cells were differentiated for a well-defined timing in an appropriate culture medium.
9
Culturing and Transfection of Human Fibroblasts and HeLa Cells
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Normal dermal human fibroblasts and dermal fibroblasts derived from a CMT2B patient were maintained in DMEM (Euroclone, ECB7501L) supplemented with 20% FBS (Euroclone, EUS0180L), 2 mM L-glutamine (Euroclone, ECB3000D), 100 units/ml penicillin-streptomycin (Euroclone, ECB3001D) at 37°C in an atmosphere of 5% CO2:air. HeLa cells were maintained as previously described.65 Briefly, they were cultured in DMEM as just described, except that 10% FBS was used. For immunofluorescence experiments cells were seeded on coverslips placed in 12-well plates; 5 × 104 HeLa cells and 5 × 103 fibroblasts were seeded in each well. For western blot and cytofluorometric analysis, 2 × 105 HeLa cells were seeded in 6-well plates and 3 × 105 fibroblast cells were seeded in 10-cm dishes. HeLa cells were transfected using Lipofectamine LTX (Life Technologies, 15338500) with the indicated plasmids (500 ng for 12-well and 1000 ng for 6-well) 24 h before harvesting for western blot analysis and 48 h before fixing for immunofluorescence.66
10
Establishment and Characterization of MeT-5A Cell Line
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The MeT-5A cell line, derived from pleural fluids obtained from non-cancerous human individuals, was purchased from the European Collection of Cell Cultures (ECACC) and cultured in RPMI 1640 medium (R0883, Sigma) plus 15% fetal bovine serum (ECS0180L, Euroclone, Milan, Italy), 100×U/mL penicillin plus 100 mg/mL streptomycin (ECB3001D, Euroclone, Milan, Italy), 2 mM L-glutamine (ECB3000D, Euroclone, Milan, Italy), and 25 mM HEPES (ECM0180D, Euroclone, Milan, Italy).
Cultures were kept in a 5% CO2–95% air humidified incubator (Napco, model 5415, Thermo Fisher Scientific, Waltham, MA, USA) at 37 °C, fed every 2–3 days, and routinely subcultured with 0.05% trypsin–2mM EDTA solution (ECM0920D, Euroclone, Milan, Italy).
Cultures were kept in a 5% CO2–95% air humidified incubator (Napco, model 5415, Thermo Fisher Scientific, Waltham, MA, USA) at 37 °C, fed every 2–3 days, and routinely subcultured with 0.05% trypsin–2mM EDTA solution (ECM0920D, Euroclone, Milan, Italy).
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