Marmoset fetal liver tissues were obtained from aborted marmoset fetus at 12–15 weeks of gestation. Fetal liver cells were isolated as previously described with minor modification15 (link). Briefly, marmoset fetal liver tissues obtained were cut into small pieces and incubated in D-Hanks’ medium containing 0.05% collagenase type IV and 5 mM CaCl2 for 30–40 min at 37 °C. Dissociated cells were collected, filtered through 70 µm sterile gauze, and centrifuged at 500 × g for 5 min. The cell pellet was resuspended and seeded on six-well plates coated with type I collagen in medium Dulbecco’s modified Eagle’s medium (DMEM)/F12 (Invitrogen, USA) supplemented with 10% fetal bovine serum, 1% penicillin/streptomycin (Invitrogen), 0.1 mM 2-mercaptoethanol (Invitrogen), 10 ng/ml hepatocyte growth factor (HGF) and EGF (Invitrogen), 1× insulin-transferrin-selenium (ITS, Invitrogen), 1 × 107 mol/l dexamethasone (Sigma, USA), and 10 ng/ml nicotinamide (Sigma). Cells were maintained at 37 °C in a 5% CO2 incubator with medium changed every other day. For passaging, confluent cultures were split 1:3 using 0.25% trypsin-EDTA for 2–5 min at 37 °C.
Insulin transferrin selenium its
Manufactured by Thermo Fisher Scientific
Sourced in United States, United Kingdom
Insulin-transferrin-selenium (ITS) is a cell culture supplement that provides essential growth factors for cell proliferation and maintenance in vitro. It contains insulin, transferrin, and selenium, which are important for various cellular processes.
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Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (7 mentions)
Nicotinamide, by Merck Group (6 mentions)
Dexamethasone (dex), by Merck Group (6 mentions)
Epidermal growth factor (egf), by Thermo Fisher Scientific (5 mentions)
Penicillin g, by Thermo Fisher Scientific (4 mentions)
Streptomycin, by Thermo Fisher Scientific (4 mentions)
Basic fibroblast growth factor (bfgf), by Thermo Fisher Scientific (4 mentions)
Hiperfect, by Qiagen (3 mentions)
Hepatocyte growth factor (hgf), by Thermo Fisher Scientific (3 mentions)
Iscove modified dulbecco media (imdm), by Thermo Fisher Scientific (3 mentions)
Tgf β1, by R&D Systems (3 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
Lipofectamine 3000 transfection kit, by Thermo Fisher Scientific (2 mentions)
Bsa fraction 5, by Merck Group (2 mentions)
Exendin 4, by OriGene (2 mentions)
B27 supplement, by STEMCELL (2 mentions)
Basic fibroblast growth factor (bfgf), by Merck Group (2 mentions)
Fetal bovine serum (fbs), by Takara Bio (2 mentions)
α mem, by Thermo Fisher Scientific (2 mentions)
Amphotericin b, by Thermo Fisher Scientific (2 mentions)
34 protocols using insulin transferrin selenium its
1
Isolation and Culture of Marmoset Fetal Liver Cells
2
Generation and Culture of Immortalized Podocyte Lines
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The generation of the human and mouse conditionally immortalized podocyte cell lines, AB8/13 and MPC-5, were previously described [10 (link),11 (link)]. AB8/13 podocytes were kindly provided by Dr. Moin A. Saleem (Bristol, UK); Dr. Peter Mundel (Charlestown, USA) kindly provided the MPC-5 cells. AB8/13 and MPC-5 cells were cultured at 33°C and the cells were differentiated in 5% CO2 incubator at 37°C for 10–15 days as indicated. The cells were cultured in RPMI 1640 medium containing 1% 100 units/ml penicillin/streptomycin and 1% Insulin-Transferrin-Selenium (ITS) (Invitrogen, Breda, the Netherlands) and 10% fetal bovine serum. For MPC-5 cells, ITS was excluded and pyruvate (1%) and interferon (10 U IFN-γ/ml) were added for cell proliferation. For differentiation of MPC-5 cells, medium without IFN-γ was used and the cells were incubated at 37°C in collagen A (10%)-coated cell culture plates (BD Biosciences, Breda, the Netherlands). The medium was refreshed at 5-day intervals. On the day of the experiment, cells were incubated in the absence (quiescent cells) or presence of TNFα (Boehringer Ingelheim GmbH, Ingelheim am Rhein, Germany) at 10 ng/ml for 24 h, unless stated otherwise.
3
Conditional Immortalization of Human Podocytes
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Conditionally immortalized human podocytes (HPCs) cell line was obtained from Dr. Moin A. Saleem (Academic Renal Unit, Southmead Hospital, Bristol, UK). HPCs were grown as described previously.16 Briefly, HPCs were cultured in RPMI‐1640 basal medium (glucose 5.5 mM, HyClone, USA) containing 10% heat‐inactivated fetal bovine serum (FBS; Gibco, USA), penicillin G (100 IU/mL), streptomycin (100 mg/mL) and 1× insulin‐transferrin‐selenium (ITS; Invitrogen, USA) at 33°C for proliferation; then they were thermoswitched to 37°C for 10–14 days without ITS to induce differentiation. All experiments were performed with differentiated cells. For high glucose (HG) stimulation, podocytes were incubated with a high concentration (30 mM) of glucose for 24 h, and mannitol (30 mM) was used as an osmotic control. For plasmid transfection, podocytes were transfected with the pEnCMV‐Nr5a2 (LRH‐1 pcDNA) plasmid and control plasmid (Miaolingbio, China) using Lipofectamine 3000 Transfection Kit (Invitrogen, USA) according to the manufacturer's instructions. For interference treatment, the small interfering RNA (siRNA) targeting GLS2 (5′‐ATCAAGATGGACTGTAA‐CAAA‐3′) was transfected into podocytes with HiPerFect (Qiagen, Germany) according to the manufacturer's instructions.
4
Redifferentiation of Human Islet Cells
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Human islets (Table 1 ) were received 2–6 days following isolation. Islets were dissociated into single cells. Cells were cultured as previously described [1 (link)] in CMRL 1066 medium containing 5.6 mM glucose and supplemented with 10% fetal bovine serum (FBS) (HyClone, Logan, UT); 100 units/ml penicillin, 100 mg/ml streptomycin, and 100 mg/ml gentamycin (PSG); 5 mg/ml amphotericin B; and 3.5 mg/ml L-glutamine. The cells were refed twice a week and split 1:2 once a week. 293T cells were cultured in DMEM supplemented with 10% FBS, PSG, and 3.5 mg/ml L-glutamine. Redifferentiation cocktail (RC), consisting of 1% BSA fraction V (Sigma), 1X insulin/transferrin/selenium (ITS, Invitrogen), D-Glucose (final concentration 25 mM), 8 nM exendin-4 (Acris), 8 nM activin A (Cytolab/PreproTech), 1X B27 supplement (Stem Cell Technologies), and 10 mM nicotinamide (Sigma), in CMRL 1066 medium supplemented with PSG, was prepared and applied to cells as previously described [27 (link)]. ALK5 inhibitor II (Enzo), and FOXO1 inhibitor AS1842856 (Millipore), were applied to cells every 48 hours at a final concentration of 0.1 μM.
5
Pharmacological Reagents for Cell Culture
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The drugs used in the present study were purchased from Sigma-Aldrich (St. Louis, MO), unless indicated otherwise: plasmocin (Fisher Scientific, Hampton, NH); EGF, dexamethasone, triiodothyronine, insulin-transferrin-selenium (ITS, 1X, Invitrogen, Carlsbad, CA); penicillin/streptomycin (1X, Invitrogen); fetal bovine serum (Invitrogen); amphotericin (Gibco Thermo Fisher Scientific, Gaithersburg, MD), and G418 (Millipore-Sigma-EMD, Burlington, MA), (−)-quinpirole hydrochloride, (S)-(−)sulpiride, LiCl, bromocriptine mesylate, L-glutamine, HEPES, sodium pyruvate, and 2-mercaptoethanol.
6
Cell-Laden Gelatin Hydrogels for Chondrogenesis
7
Podocyte Culture and Manipulation
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The conditionally immortalised human podocytes (HPCs) cell line was provided by Dr. Moin A. Saleem (Academic Renal Unit, Southmead Hospital, Bristol, UK). Podocytes were grown in RPMI culture medium (HyClone) containing 10% heat‐inactivated fetal bovine serum (FBS; Gibco), 100 U/ml penicillin G, 100 μg/ml streptomycin (Invitrogen), and 1× insulin–transferrin–selenium (ITS; Invitrogen) at 33°C for proliferation, then were shifted to 37°C for 14 days without ITS for differentiation. For Ang II stimulation, podocytes were treated with Ang II (10−7 M) for 24 h. For lysosome inhibition, podocytes were incubated with Leupeptin (20 mΜ, Topscience), an inhibitor of lysosomal protein degradation, for 30 min. For knockdown treatment, small interfering RNAs (siRNAs) targeting Rab11 (Qiagen) were transfected with HiPerFect (Qiagen) according to the manufacturer's instructions. For plasmid transfection, podocytes were transfected with the pEGFP‐Rab11a‐WT plasmid (Miaolingbio) using Lipofectamine 3000 Transfection Kit (Invitrogen) according to the manufacturer's instructions.
8
Hepatocyte-like Cell Differentiation Protocol
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ADHLSCs at passage 5 or 6 were seeded at a density of 1 × 104 cells/cm2 onto collagen I-coated 175 cm2 flasks in complete DMEM medium. Twenty-four hours later, the culture medium was switched to IMDM (Thermo Fisher Scientific). Cells were then subjected to a four-step differentiation protocol as previously described [39 (link)]. First, cells were incubated for 2 days with IMDM containing 20 ng/ml epidermal growth factor (EGF) (PeproTech) and 10 ng/ml basic fibroblast growth factor (bFGF) (PeproTech). Then, the cells were incubated for 10 days with IMDM containing 20 ng/ml hepatocyte growth factor (HGF) (PeproTech), 10 ng/ml bFGF, nicotinamide 0.61 g/l (Sigma Aldrich), and 1% insulin-transferrin-selenium (ITS) (Invitrogen) premix. Next, the cells were incubated for 10 days with IMDM containing 20 ng/ml HGF, 20 ng/ml oncostatin M (OSM) (PeproTech), 0.61 g/l nicotinamide, and 1% ITS premix. Finally, the cells were treated with IMDM containing 20 ng/ml OSM, 1 μM dexamethasone (Sigma Aldrich), and 1% ITS premix for 10 days. For each step, the medium was changed every three days. Negative controls were performed using IMDM supplemented with 1% FCS and 1% of Penicillin/Streptomycin. Cells were harvested either after each step or at the end of the differentiation protocol and used for MLR, RT-PCR, fluorescence microscopy, or flow cytometry analysis.
9
Directed Redifferentiation of Islet Cells
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Expanded human islet cells or sorted BCD cells in passages 5–7 were infected with β-catenin shRNA or nontarget shRNA viruses. Five to 6 days following infection cells were trypsinized, pelleted, and seeded at 3.8×104 cells/cm2 in ultra-low attachment plates (Corning) in CMRL 1066 medium containing 5.6 mM glucose and supplemented with 100 U/ml penicillin, 100 µg/ml streptomycin, 100 µg/ml gentamycin sulphate (Biological Industries), 1% BSA fraction V (Sigma), 1× insulin/transferrin/selenium (ITS, Invitrogen), D-Glucose (final concentration 25 mM), 8 nM exendin-4 (Acris), 8 nM activin A (Cytolab/PreproTech), 1× B27 supplement (Stem Cell Technologies), and 10 mM nicotinamide (Sigma) (Redifferentiation Cocktail, RC) for the indicated periods. Half-volume medium changes were performed every other day.
10
Epicardial Cell Lines for Hypoxia Studies
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Immortalized epicardial cell lines, isolated from 13.5 dpc Sm22α-lacZ mice, crossed with the ImmortoMouse line were maintained at 33 °C in DMEM containing 10% FBS (Atlanta Biologicals, Flowery Branch, GA, USA), insulin-transferrin-selenium (ITS; Invitrogen, Grand Island, NY, USA) and 10 units/mL mouse gamma interferon (Peprotech, Rocky Hill, NJ, USA). Experimental cells were transferred to 5% FBS DMEM medium and cultured at 37 °C as previously described [21 (link)]. Hypoxia was achieved by culturing cells in 1% oxygen conditions.
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