MEFs, PANC-1, A549, A375, A549/NF-κB-Luc and HeLa/NF-κB-Luc cells were cultured in Gibco high glucose DMEM (Life Technologies) with 10% FBS. G-361 cells were cultured in McCoy’s 5A medium (Life Technologies) with 10% FBS. All other cell lines were cultured in RPMI 1640 medium with 10% FBS. Culture media for all cell lines was supplemented with 1% penicillin/streptomycin. Media for A549/NF-κB-Luc and HeLa/NF-κB-Luc cells also contained 0.1 mg/mL hygromycin-B. NHDF and NHLF cells were cultured in fibroblast basal medium (FBM) supplemented with 0.1% insulin, 0.1% rhFGF-B, 0.1% GA-1000, and 2% fetal bovine serum (Lonza). NHMCs were cultured in mesangial cell basal growth medium (MsBM) supplemented with 5% fetal bovine serum and 0.1% GA-1000 (Lonza). Cells were cultured in a humidified atmosphere at 37°C with 5% CO2. RTA 408 and bardoxolone methyl were dissolved in DMSO (vehicle). The final amount of DMSO in the media was ≤ 0.1% and was equivalent in drug- and vehicle-treated samples.
Ga 1000
Manufactured by Lonza
Sourced in Switzerland, United States, Belgium
The GA-1000 is a laboratory instrument designed for the analysis and quantification of various chemical and biological compounds. It utilizes advanced spectroscopic techniques to provide accurate and reliable measurements. The core function of the GA-1000 is to enable precise analytical testing and data generation in a laboratory setting.
Automatically generated - may contain errors
Lab products found in correlation
Human epidermal growth factor (hegf), by Lonza (33 mentions)
Ascorbic acid, by Lonza (20 mentions)
Hydrocortisone, by Lonza (20 mentions)
Vascular endothelial growth factor (vegf), by Lonza (20 mentions)
Insulin, by Lonza (18 mentions)
Fetal bovine serum (fbs), by Lonza (18 mentions)
Hfgf b, by Lonza (17 mentions)
R3 igf 1, by Lonza (13 mentions)
Heparin, by Lonza (12 mentions)
L glutamine, by Lonza (7 mentions)
Mda mb 231, by American Type Culture Collection (7 mentions)
Mcf 10a, by Lonza (6 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (6 mentions)
Trypsin neutralizing solution, by Lonza (5 mentions)
Egm 2, by Lonza (5 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (5 mentions)
Mcf 7, by American Type Culture Collection (5 mentions)
Mcf 10a, by American Type Culture Collection (5 mentions)
Penicillin, by Thermo Fisher Scientific (5 mentions)
Streptomycin, by Thermo Fisher Scientific (5 mentions)
72 protocols using ga 1000
1
Cell Culture Protocols for Diverse Cell Lines
2
Adipogenic Differentiation of hMSCs with SDC-1 Knockdown
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hMSC populations (n = 2) were plated at 2.1 × 104 cells/cm2 onto 24-well plates in MSCGM™ in technical triplicates. The adipogenesis protocol was provided by the manufacturer (Lonza, Walkersville, MD, USA, Document #AA-2501-16 07/11). Once cells reached 90%-95% confluency, adipogenesis was induced by replacing the MSCGM™ with Adipogenesis Induction Medium (AIM) supplemented with SingleQuots™ of h-insulin (recombinant), L-glutamine, MCGS, dexamethasone, indomethacin, IBMX (3-isobutyl-l-methyl-xanthine) and GA-1000 (Lonza, Walkersville, MD, USA). After one day of adipogenesis, SDC-1 knockdown (SDC-1 KDAD) was induced by introducing siRNA in low serum (2%) AIM, along with no siRNA control (Untreated, UTAD) and non-targeting siRNA control (scrambled; SCRAD). hMSC adipogenic cultures were incubated with siRNA for 96 h and the medium replaced with Adipogenesis Maintenance Medium (AMM) consisting of SingleQuots™ of h-insulin (recombinant), L-glutamine, MCGS and GA-1000 (Lonza, Walkersville, MD, USA) for 2 days. hMSC cultures then underwent two more cycles of AIM (3 days) and AMM (2 days) followed by maintenance in AMM for 7 days prior to termination of differentiation (22 days in total). hMSC adipogenic cultures under SDC-1 knockdown conditions were harvested for RNA and protein, as well as stained for Oil Red O.
3
Oncosphere Formation Assay for Tumor Initiation
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To assess tumor initiation capacity in vitro, cells were counted and plated into low-attachment 96-well plates at dilution of 1 cell per well. They were then cultured in mammary epithelial basal medium (MEBM, Lonza, cat no. CC-3151), supplemented with MEGM SingleQuots (which contain Insulin, EGF, Hydrocortisone and GA-1000, LONZA cat no. 4136), 1X B27 without retinoic acid (GIBCO, cat no. 12787-010), and 20 ng/ml of recombinant fibroblast growth factor (GIBCO, cat no. PHG0026), and incubated in 5% CO2, 37°C in order to obtain a first generation of oncospheres (anoikis and pluripotency selection) after 15 days. The process was repeated to ensure second-generation oncospheres (pluripotency selection). After 2 weeks of culture, the oncospheres were counted under the microscope.
4
Pericyte Adipogenic Differentiation Protocol
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Pericytes were seeded in 24-well plates at 4 × 104 cells/well and cultured with Mesenchymal Stem Cell Basal Medium (MSCBM, Lonza, Basel, Switzerland, #PT-3238) until the cells became 100% confluent. After reaching confluence, the medium was changed to adipogenic induction medium (Lonza, #PT-3102B) supplemented with h-insulin, L-glutamine, mesenchymal cell growth supplement (MCGS), dexamethasone, indomethacin, isobutylmethylxanthine (IBMX), and GA-1000 (Lonza, #PT-4135) for 4 days. Then, the cells were cultured in adipogenic maintenance medium (Lonza, #PT-3102A) supplemented with h-insulin, L-glutamine, MCGS, and GA-1000 (Lonza, #PT-4122) for 3 days. The above processes of induction for 4 days and maintenance for 3 days were repeated 3 times to stimulate adipogenic differentiation. Noninduced control cells were cultured with only the supplemented adipogenic maintenance medium on the same schedule. To evaluate the adipogenesis of pericytes, quantitative PCR analysis and Oil Red O staining were performed.
5
Isolation and Culture of Human Aortic Valve Endothelial Cells
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Human aortic valves from the patients were collected in a cold, sterile saline solution. After incubation with collagenase II solution [600 U/mL in EBM-2 Endothelial Cell Growth Basal Medium-2 (Lonza, #CC-3156) with 10% FBS and GA-1000 (1:1000, Lonza, # CC-4083)] for 10 min at 37 °C, human aortic VECs were isolated from the aortic valve tissue, by a gentle rolling of pre-soaked sterile cotton swaps, according to a previous report by Gould & Butcher70 . Isolated VECs were cultured in EGM-2 Endothelial Cell Growth Medium-2 BulletKit (Lonza, #CC-3162), supplemented with 10% FBS in a humidified CO2 incubator (5% CO2, 37 °C). When 90 % confluency was reached, cells were trypsinized and then subcultured, at a 1:3 ratio. Gelatin-coated cell culture flasks (T25 for passage 0 and T75 for passage 1 or higher) were used for cell culture. VECs at passage 1-2 were used for further experiments.
6
Cultivation of Human Glioblastoma Cell Lines
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A total of six human glioblastoma (GBM) cell lines, including U87MG [GBM of unknown origin; American Type Culture Collection HTB-14; short-tandem repeat (STR) profiling was performed], LN229, H4, U251, U118 (derived from the U138MG astrocytoma cell line; American Type Culture Collection HTB-15; STR profiling was performed) and A172, were obtained from The Cell Bank of Type Culture Collection of The Chinese Academy of Sciences. The human 293T cell line was obtained from American Type Culture Collection. All cells were sustained in DMEM (HyClone; Cytiva) supplemented with 10% FBS (HyClone; Cytiva), 100 U/ml penicillin and 100 ng/ml streptomycin. Normal human astrocytes (NHAs) were purchased from Lonza Group, Ltd. and cultured in the provided astrocyte growth media (Lonza Group, Ltd.) supplemented with 0.1% recombinant human epidermal growth factor, 0.25% insulin, 0.1% ascorbic acid, 0.1% GA-1000, 1% L-glutamine (all Lonza Group, Ltd.) and 5% FBS. All cells were cultured at 37°C in a humidified atmosphere with 5% CO2.
7
Culturing Human Umbilical Vein Endothelial Cells
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Human umbilical vein endothelial cells (HUVECs) were purchased from Lonza and cultured in endothelial basal medium-2 (EBM-2) supplemented with EGM-2MV SingleQuot additives (fetal bovine serum (5% FBS), hEGF, hydrocortisone, VEGF, hFGF-B, R3-IGF-1, ascorbic acid, GA-1000, and heparin; Lonza). Murine NIH-3T3 fibroblasts were maintained in high glucose, sodium pyruvate and GlutaMAX-supplemented Dulbecco’s modified Eagle medium (DMEM; Gibco) with 10% FBS (Biosera) and 1% Penicillin/Streptomycin (Gibco). Cells were incubated at 37 ℃ in a humidified atmosphere containing 5% CO2.
8
Culturing human brain endothelial cells
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Human brain endothelial cells (hCMEC/D3 cell line) were cultured as previously described [32 (link)]. Briefly, cells were cultured in filtered EBM-2 basal medium supplemented with FBS, hydrocortisone, hFGF-B, VEGF, R3-IGF-1, ascorbic acid, hEGF, and GA-1000 (CC-3202, Lonza Bioscience, Basel, Switzerland) and used for experiments at passages 23–35. The medium was changed every 2–3 days, and the cells were passaged every 4–5 days when they reached 90% confluence. Cells were kept on culture ware coated with 5 μg/cm2 rat tail collagen I (Cat no. 122-20, Sigma-Aldrich Merck, Darmstadt, Germany). For all assays (RNA analysis, cell viability, and permeability assays), cells were plated at 50,000 cells per cm2 in growth factor-negative medium (EBM-2 with 10 mM HEPES supplemented with FBS, bFGF, hydrocortisone, and ascorbic acid) for 6 days to allow the growth of a confluent monolayer yielding maximum barrier formation.
9
Quantification of EPHB2-Mediated Sphere Formation
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Cells were seeded in ultra-low attachment surface 24-well plates (Corning Incorporated, Corning, NY, USA) at a density of 4×103 cells per well, with MEGM Bullet kit serum-free medium supplemented with 0.4% BPE, 0.1% hEGF, 0.1% hydrocortisone, 0.1% GA1000, 0.1% insulin, 1% l-glutamine (Lonza, Walkersville, MD, USA). To examine the effect of EPHB2 on sphere formation efficiency, cells were immediately transfected with control siRNA or EPHB2 siRNA. Five days after seeding, the number of spheres >100 µm in diameter in eight microscopic fields at ×50 magnification was counted. Representative images were photographed at ×200 magnification.
10
Sodium Iodate-Induced Retinal Pigment Epithelial Cell Signaling
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Sodium Iodate (NaIO3; 71702) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Retinal Pigment Epithelial Cell Growth Medium (RtEGMTM; #00195407) with supplements including 2% FBS, 2% L-glutamine, 0.5% bFGF, 0.1% GA-1000 was purchased from LONZA (Walkersville, MD, USA). Dulbecco’s modified Eagle’s medium (DMEM; 12800-017), fetal bovine serum (FBS; 26140-079), penicillin/streptomycin (10378-016), and 0.25% trypsin (25200-072) and other cell culture reagents were purchased from Gibco (Gaithersburg, MD, USA). Primary antibodies including phosphor-Akt (ser473; #4058, Thr308; #9275), total Akt (#9272), phospho-ERK (#4370), ERK (#4695), phosphor-JNK (#9251), JNK (#9252), phosphor-p38 (#4511), p38 (#9212), phosphor-IκBα (#9246), and IκBα (#9242) (Cell Signaling Technology, Inc., Danvers, MA, USA), and β-actin polyclonal antibody (SC-47778) (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) were used for western blotting analysis. Human pentraxin 3 (PTX3; DY1826) ELISA kit was purchased from R&D System, Inc. (Minneapolis, MN, USA). U0126 (BML-EI282), LY194002 (BML-ST420), SP600125 (BML-EI305), SB203580 (BML-EI286), BAY 11-7082 (BML-EI278) (Enzo Life Sciences, Farmingdale, NY, USA), and NAC (A7250) (Sigma-Aldrich, St. Louis, MO, USA) were used for inhibitor reagents.
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