Leishmania donovani 1S promastigotes were cultured as earlier [22 (link)] in medium M199 (Lonza, Switzerland) supplemented with fetal bovine serum (10%; Invitrogen), hemin and adenine (Sigma Aldrich, USA). Isolation of whole cell extracts, analysis of growth patterns and generation time, synchronization regimes and flow cytometry analysis methods, transfections of Leishmania promastigotes, and creation and maintenance of clonal lines were done as described in Supplementary Methods (Supporting Information).
M199 medium
Manufactured by Lonza
Sourced in United States, Switzerland, Spain, Belgium
M199 medium is a cell culture medium that provides essential nutrients and growth factors for the cultivation of a variety of cell types. It is a complex mixture of amino acids, vitamins, salts, and other components that support cell growth and proliferation. The core function of M199 medium is to maintain the viability and optimal growth conditions for cultured cells.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (5 mentions)
L glutamine, by Lonza (5 mentions)
Penicillin, by Lonza (5 mentions)
Streptomycin, by Lonza (5 mentions)
Fetal calf serum (fcs), by Merck Group (4 mentions)
Pen strep, by Lonza (3 mentions)
L glutamine, by Merck Group (3 mentions)
Fibrinogen, by Enzyme Research (3 mentions)
Hemin, by Merck Group (2 mentions)
Adenine, by Merck Group (2 mentions)
Fetal bovine serum (fbs), by Merck Group (2 mentions)
Ecgs heparin, by PromoCell (2 mentions)
Gelatin, by Merck Group (2 mentions)
Heat inactivated newborn calf serum, by Thermo Fisher Scientific (2 mentions)
Crude endothelial cell growth factor, by Lonza (2 mentions)
Endothelial cell growth supplement (ecgs), by PromoCell (2 mentions)
Penicillin, by Merck Group (2 mentions)
Streptomycin, by Thermo Fisher Scientific (2 mentions)
Heparin, by Merck Group (2 mentions)
Penicillin g, by Thermo Fisher Scientific (2 mentions)
30 protocols using m199 medium
1
Culturing and Analyzing Leishmania donovani
2
Fabrication of Collagen-Based Spiral Vessels
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Collagen gels were prepared by mixing an acidic collagen stock solution (at 15 mg/ml) with 1 N NaOH, 10× Medium M199, and endothelial growth medium (Lonza) to reach neutralization and targeted concentration (6 or 7.5 mg/ml). After homogenizing, the collagen mixture was degassed by placing under vacuum for 30 to 60 min. While the gel solution was under vacuum, spiral vessel holders were prepped by brief corona treatment (<30 s) and subsequent coating of polyethylenimine (1%; Sigma-Aldrich) with glutaraldehyde (0.1%; Sigma-Aldrich) cross-linking. After five washes with autoclaved water, vessel holders were dried and placed in the spiral retractor system where the spiral pattern was brought into contact with a 25 mm length of 23-gauge hypodermic tubing within the holder. The degassed gel mixture was then pipetted into the reservoir and allowed to cross-link at room temperature for 1 hour. After cross-linking, the spiral pattern was removed from the gel using the spiral retraction system and submerged in warm medium. Each spiral vessel was seeded with cells following the same protocol as in PDMS vessels.
3
Cell Culture Conditions for U87, HMEC-1 and GSCs
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U87 cells were maintained in DMEM/F-12 (Lonza, Verviers, Belgium) supplemented with 1% fetal bovine serum (FBS, Sigma-Aldrich, Munich, Germany) and 1% penicillin/streptomycin solution (pen/strep, Lonza). Human dermal microvascular endothelial cells (HMEC-1) were kindly provided by Dr. E.W. Ades (CDC, Atlanta, GA, USA) [49 (link)] via Prof. G. Molema and the UMCG Endothelial Cell Facility and maintained in M-199 medium (Lonza) supplemented with 10% FBS, 10% human serum (Sigma-Aldrich), pen/strep and L-glutamine (Lonza). GSCs were maintained in DMEM/F12 (Lonza) supplemented with 10% B27 (Life Technologies, Bleiswijk, the Netherlands), 20 ng/ml bFGF and EGF (Life Technologies) and pen/strep. Carrier-free recombinant human VEGFA165 (from here on referred to as VEGFA), Ang-2 and IL-8 were obtained commercially (R&D Systems, Minneapolis, MN, USA).
4
Isolation and Culture of HUVECs
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Human umbilical vein endothelial cells (HUVEC) were isolated from umbilical cords using collagenase treatment essentially as described (Jaffe et al., 1973 (link); Zhang et al., 1997 (link)). The studies were reviewed and approved by Ethics Commission of the Medical University of Vienna. Written informed consent was provided by the participants’ legal next of kin. Cells were seeded in 75 cm2 flasks coated with 1% gelatin (Sigma, St. Louis, MO, United States, #04055) and cultured in M199 medium (Lonza, Basel, Switzerland, #12-119F) with 20% heat-inactivated FBS (Sigma, St. Louis, MO, United States, #F6765), penicillin (100 units/ml), streptomycin (100 μg/ml), (Pen-Strep, Lonza, Basel, Switzerland, #DE17-602E), 2 mM L-glutamine (Sigma; #G7513), 5 units/ml heparin, and 25 μg/ml ECGS (Promocell, Heidelberg, Germany, ECGS/Heparin #C-30140). Cells were passaged at a ratio of 1:3 and used until passage 5 for experiments.
5
Isolation of Human Umbilical Vein Endothelial Cells
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Human umbilical vein endothelial cells (HUVECs) were isolated from umbilical cords from healthy donors obtained from the Amstelland Ziekenhuis, Amstelveen. The use of human tissue for isolation of endothelial cells was approved by the Medical Ethical Committee of the VU University Medical Center. Patients gave informed consent for the use of tissue for research purposes. The study conforms with the Declaration of Helsinki. After isolation, cells were resuspended in M199 medium (Biowhittaker/Lonza), supplemented with penicillin 100 U/mL and streptomycin 100 μg/mL (Biowhittaker/Lonza), heat‐inactivated human serum 10% (Sanquin Blood Supply, Amsterdam, The Netherlands), heat‐inactivated newborn calf serum 10% (Gibco, Grand Island, NY), crude endothelial cell growth factor 150 μg/mL (prepared from bovine brains), L‐glutamine 2 mmol/L (Biowhittaker/Lonza), and heparin 5 U/mL (Leo Pharmaceutical Products, Weesp, The Netherlands). Cells were cultured at 37 ⁰C and 5% CO2, with a change of culture medium every 2 days. Cells were cultured up to passage 2; for experiments, passage 1 to 2 cells were used.
6
Cultivating HUVEC Monolayer on Transwell
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Human umbilical vascular endothelial cells (HUVECs) were seeded on the inner chamber of collagen-coated Transwell filters (0.33-cm2, Corning Life Sciences) in M199 medium (Biowhittaker) supplemented with 2 mM L-glutamine, 10% FBS, 10μg/ml endothelium mitogen (Fisher), 20μg/ml heparin sodium salt (Sigma) and 100 U penicillin/streptomycin following a previously described protocol [68 (link)]. The cell monolayer was allowed to form over 4–5 days.
7
Isolation and Culture of HUVECs
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The study was executed in accordance with the Declaration of Helsinki and was approved by the University Human Subjects Committee of the VU University Medical Center. For isolation of Human Umbilical Vein Endothelial Cells (HUVECs), umbilical cords from healthy donors were obtained from the Amstelland Ziekenhuis, Amstelveen. Cells were isolated from umbilical cord veins according to the protocol of Jaffe.29 Cells were cultured in 1% gelatin coated plates (Costar) with M199 medium containing 2 mMol/L L -glutamine, 100 U/mL penicillin, 100 mg/mL streptomycin (all Lonza) 10% heat-inactivated New Born Calf Serum from (Gibco), 10% heat-inactivated Human serum (Sanquin CLB), 5 U/mL heparin (Leo Pharmaceuticals products) and crude endothelial cell growth factor prepared from bovine brains. Cells were kept in incubators with 5% CO2 at 37 °C, with a medium change every other day. Confluent cells were washed with M199, trypsinized (0.05% trypsin, Gibco) and seeded in a 1:3 dilution. Passage 1 or 2 cells consisting of pools from 3 donors were used unless indicated otherwise, in all experiments.
8
Cultivation and Characterization of Leishmania donovani Cells
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Leishmania donovani 1S cells were maintained at 26°C in M199 medium (Lonza) supplemented with 10% fetal bovine serum (Invitrogen), adenine, glutamine and hemin (all from Sigma Aldrich, USA), as described earlier [40 (link)]. Details of isolation and fractionation of cell extracts, growth and survival analyses, determination of generation time, synchronization regimes, flow cytometry regimes are given in S1 Methods .
9
Isolation and Adipogenesis of SAT and EAT
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After washing the fat pads three times, the stromal vascular cells (SVC) from consecutive SAT and EAT of 17 patients were isolated and cultured following the collagenase digestion protocol [5 (link)]. Then, cells were or were not induced to adipogenesis with M199 medium (Lonza Biologics, Porriño, Spain) supplemented with 10% foetal bovine serum (FBS), and the adipogenesis cocktail, composed of 5 μg/mL insulin, 250 nM dexamethasone, 0.5 mM methylisobutylxanthine and 1 μM thiazolidinedione (IDMT). All pharmacological drugs were obtained from Merck Life Science S.L.U. (Madrid, Spain). In dedifferentiated epicardial or subcutaneous adipocytes from 4 patients, we performed an adipogenesis treatment with IMT (insulin, methylisobutylxanthine, thiazolidinedione) supplemented or not with aldosterone (1 µM) and/or mineralocorticoid receptor antagonist (MRA) (spironolactone 5 µM).
10
Isolation and Culture of Human Peritoneal Mesothelial Cells
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We isolated HPMCs from human omentum as described previously.18 (link) Harvesting of the omentum was permitted by the Medical Ethics Committee of Hiroshima Graduate School of Biomedical Science (E-2161). Written Informed consent was obtained from each patient. HPMCs were maintained in M199 medium (Lonza, Basel, Switzerland) containing 10% fetal bovine serum and penicillin-streptomycin. The cells were seeded into 6-well plates. At subconfluence, HPMCs were incubated for 24 hours in M199 medium and then treated with 4.25% high glucose or identical concentrations of Mannitol for up to 48 hours. Mannitol was purchased from Fujifilm wako chemicals (Osaka, Japan) and glucose solution was purchased from Sigma-Aldrich (St. Louis, MO, USA).
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