SV40LT-immortalized MEFs were grown in D-MEM (Lonza, BE12-614F) supplemented with 10% fetal bovine serum (EuroClone, ECS0180L), 2 mM L-glutamine (EuroClone, LOBE17605F), 100 U/ml penicillin-0.1 µg/ml streptomycin (EuroClone, ECB3001L), 0.1 mM non-essential amino acids (Microtech, X-0557). U2OS cells (ATCC) were grown in Mc Coy’s 5 A w/Glutamax (Life Technologies, 36600-088) supplemented with 10% fetal bovine serum (EuroClone, ECS0180L). U2OS cells were authenticated using the GenePrint® 10 System (10-Locus STR System for Cell Line Authentication) by Promega CAT. NUM. B9510. HeLa 1.3, HeLa204 and HTC75 cells were grown in D-MEM (Lonza, BE12-614F) supplemented with 10% fetal bovine serum (EuroClone, ECS0180L), 2 mM L-glutamine (EuroClone, LOBE17605F), 100 U/ml penicillin-0.1 µg/ml streptomycin (EuroClone, ECB3001L). HeLa1,3, HeLa204, and HTC75 cell lines are a gift from Titia de Lange. All cell lines are tested for mycoplasma both upon arrival at IFOM and after a new stock of cells is made, and all of them resulted to be negative for mycoplasma contamination. Mycoplasma test is performed by the IFOM Cell Biology UNIT and consist of two independent tests: a PCR analysis (For detail protocol see36 (link) and a biochemical test (MycoAlert Detection Kit, Lonza Catalog #: LT07-418).
Ecs0180l
Manufactured by Euroclone
Sourced in Italy
The ECS0180L is a laboratory equipment product from Euroclone. It is designed to perform specific functions within a laboratory setting. The core function of this product is to enable laboratory procedures, but no further details about its intended use or capabilities can be provided in an unbiased and factual manner.
Automatically generated - may contain errors
Lab products found in correlation
Ecb3001d, by Euroclone (12 mentions)
Ecb3000d, by Euroclone (9 mentions)
G8644, by Merck Group (3 mentions)
Ecb7501l, by Euroclone (3 mentions)
Rpmi 1640 medium, by Merck Group (3 mentions)
Ecm0728l, by Euroclone (3 mentions)
Penicillin streptomycin, by Merck Group (3 mentions)
Ecb2071l, by Euroclone (2 mentions)
Dulbecco modified eagle medium (dmem), by Merck Group (2 mentions)
Ecm0749l, by Euroclone (2 mentions)
S8022, by Selleck Chemicals (2 mentions)
Bi 10773, by Selleck Chemicals (2 mentions)
Fetal bovine serum (fbs), by Euroclone (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Flp in 293 trex cells, by Thermo Fisher Scientific (1 mentions)
Penicillin, by Merck Group (1 mentions)
Streptomycin, by Merck Group (1 mentions)
Zeocin, by InvivoGen (1 mentions)
Blasticidin, by InvivoGen (1 mentions)
Tirzepatide, by Selleck Chemicals (1 mentions)
27 protocols using ecs0180l
1
Cell Culture and Authentication Protocol
2
Differentiation of Human Cardiac AC16 Cells
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The human cardiac myocyte AC16 cell line was obtained from Merck (Master cell bank passage 4, Lot: RD1606008; SCC109). All experiments were performed within 10 passages of the working cell bank. For cultivation, the cells were kept in growth medium (DMEM/Nutrient Mixture F-12 (04-687F/U1, Lonza), 12.5 V/V% FBS (ECS0180L, EuroClone or 35-079-CV, Corning, Corning, NY, USA), 100 uU/mL penicillin, 100 ug/mL streptomycin, and 25 ng/mL amphotericin B (30-004-CI, Corning)) at 37 °C in a humidified atmosphere of 5% CO2. Prior to dividing the subcultures, cells were maintained until 70% confluence. For differentiation of AC16 cells, growth medium was changed to differentiation medium with 2% FBS supplemented with 10 nM ATRA and 1× insulin-transferrin-selenite supplement (DMEM/Nutrient Mixture F-12 (04-687F/U1, Lonza), as well as 2 V/V% FBS (ECS0180L, EuroClone or 35-079-CV, Corning), 100 uU/mL penicillin, 100 ug/mL streptomycin and 25 ng/mL amphotericin B (30-004-CI, Corning), 1× ITS (I3146, Sigma), and 10 nM ATRA (R2625, Sigma)) [63 (link)]. AC16 and diffAC16s were seeded on 96-well plates with 2 × 104 and 1 × 104 seeding density, respectively.
3
Cultivation of Common Cell Lines
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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.
4
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.
5
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).
6
Cellular Starvation and Genetic Manipulation
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All cells were cultured in DMEM-High glucose (cat. no. ECM0728L, Euroclone) supplemented with 10% FBS (cat. no. ECS0180L, Euroclone), penicillin (100 IU/ml), and streptomycin (100 μg/ml) (cat. no. ECB3001D, Euroclone). For the starvation treatments, cells were washed twice using PBS Ca2+/Mg++ free, and HBSS (14025092, Gibco) supplemented with HEPES (H0887, Euroclone) was added for the indicated time points.
HeLa cells were obtained from ATCC. HeLa-FLCN KO cells were a kind gift of Z. P. Arany. UOK-257 cells were obtained from W. Marston Linehan, MD (The National Cancer Institute, Bethesda). All cell culture incubations were performed at 37 degrees with 5% CO2.
cDNA sequences for overexpression of EGR1 and TFEB were cloned into pLVX-EF1a-PURO and pLVX-TetOne-Puro (Clontech) lentiviral backbones, respectively. For RNA interference, shRNA sequences against EGR1 were cloned in the pLKO.1_hPGK-Puro-CMV-tGFP lentiviral backbone (Sigma-Aldrich).
HeLa cells were obtained from ATCC. HeLa-FLCN KO cells were a kind gift of Z. P. Arany. UOK-257 cells were obtained from W. Marston Linehan, MD (The National Cancer Institute, Bethesda). All cell culture incubations were performed at 37 degrees with 5% CO2.
cDNA sequences for overexpression of EGR1 and TFEB were cloned into pLVX-EF1a-PURO and pLVX-TetOne-Puro (Clontech) lentiviral backbones, respectively. For RNA interference, shRNA sequences against EGR1 were cloned in the pLKO.1_hPGK-Puro-CMV-tGFP lentiviral backbone (Sigma-Aldrich).
7
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.
8
Cell Line Cultivation and Authentication
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Hep3B and HepG2 cell lines were purchased from the Leibniz-Institut DSMZ, Braunschweig (ACC-93, ACC-180) and grown in DMEM (Euroclone, ECM0749L) and RPM1640 (Euroclone, ECB9006L), respectively, supplemented with 10% FBS (Euroclone, ECS0180L) and 1% penicillin-streptomycin. HuS cells were a gift of Dr. Vinicio Carloni, University of Florence and were grown in DMEM containing 4.5 g/L of glucose (Euroclone, ECB7501L) and supplemented with 10% FBS (Euroclone, ECS0180L), 5 ng/mL EGF (Sigma-Aldrich, E9644), 420 ng/mL insulin (Sigma-Aldrich, I9278), 20 ng/mL selenium (Sigma-Aldrich, S9133), 1% DMSO (Amresco, 0231) and 1% penicillin-streptomycin (Euroclone, ECB3001D). Cells have been authenticated by STR PCR by the supplier and routinely tested for Mycoplasma contamination using Myco Alert (Lonza, LT07-318).
9
Wound Healing Assay with Serum Starvation
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Cells were transfected as required and let grow until confluent. Then, cells were serum-starved overnight to prevent proliferation, medium replaced with one containing 1% FBS (Euroclone, ECS0180L) and a wound created using a pipette tip. Images were taken using an inverted microscope connected with a digital camera at the beginning (T0) and end of the experimental time (T12-24h). Quantification of gap closure was performed using the ImageJ software. In selected experiments inhibitors were added 1 h before T0.
10
HeLa Cell Transfection for Live FLIM-FRET
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HeLa cells were maintained in DMEM (Euroclone, cod. ECM0060L) medium with 10% fetal bovine serum (Euroclone, ECS0180L), 100 U/ml penicillin, 100 mg/ml streptomycin sulphate (Euroclone, cod. ECB3001D) and 2 mM L-Glutamine (Euroclone, cod. ECB3000D-20) at 37 °C in an atmosphere of 5% CO2 and 95% humidity. Cells seeded at an 70% confluent onto 6-well plates were transiently transfected with PRK-RFP-exon1 HTT with 20Q (kindly provided from S. H. Li Laboratory, Emory University)35 (link) or pEGFP-exon 1 HTT with 74Q (Addgene, 40262) or cotrasfected with PRK-RFP-exon1 HTT with 20Q and pEGFP-exon 1 HTT with 74Q plasmids using Xfect transfection reagent (Clontech, cod 631317). After 48 h from transfection cells were used for live FLIM-FRET analysis.
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