Human EBV transformed lymphoblastoid cell lines, GM06990 (control) and GM03200 (Fragile X), and fibroblast cell lines, GM00357 (control) and GM05848 (Fragile X), were purchased from Corielle institute. Lymphoblastoids were grown in RPMI1640 (Corning), supplemented with GlutaMAX (GIBCO), 15% heat-inactivated FBS (Fetal Bovine Serum, Benchmark), 100 IU/mL penicillin and 100 μg/mL streptomycin (Corning) at 37°C with 5% CO2. Fibroblast cells were cultured in MEM culture media with 15% FBS (Corning), 1X GlutaMAX, 100 IU/mL penicillin and 100 μg/mL streptomycin. Cell lines were verified for presence or absence of FMRP and expansion of the FMR1 5′UTR using western blot and Southern blot respectively. See Figures 1A and S1A for results. Phoenix-AMPHO producer cells (ATCC) were grown in DMEM medium (GIBCO) supplemented with 10% FBS, 1X GlutaMAX, 100 IU/mL penicillin and 100 μg/mL streptomycin, 1mM sodium pyruvate (Corning), 10mM HEPES buffer (Corning) and 1X MEM non-essential amino acids (Corning).
Glutamax
Manufactured by Corning
Sourced in United States
GlutaMAX is a laboratory product manufactured by Corning. It serves as a substitute for L-glutamine in cell culture media. GlutaMAX provides a stable source of glutamine for supporting cell growth and proliferation in vitro.
Automatically generated - may contain errors
Lab products found in correlation
Glutamax, by Thermo Fisher Scientific (19 mentions)
Streptomycin, by Corning (7 mentions)
Penicillin streptomycin, by Corning (6 mentions)
Sodium pyruvate, by Corning (5 mentions)
Rpmi 1640, by Corning (5 mentions)
Penicillin, by Corning (5 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (5 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (4 mentions)
Streptomycin, by Thermo Fisher Scientific (4 mentions)
Mda mb 231, by American Type Culture Collection (3 mentions)
Fetal bovine serum (fbs), by Merck Group (3 mentions)
Fetal bovine serum (fbs), by Corning (3 mentions)
Epidermal growth factor (egf), by Thermo Fisher Scientific (3 mentions)
Trypsin edta, by Thermo Fisher Scientific (3 mentions)
Non essential amino acids (neaa), by Corning (2 mentions)
Non essential amino acids (neaa), by Thermo Fisher Scientific (2 mentions)
β mercaptoethanol, by Thermo Fisher Scientific (2 mentions)
Hepes, by Corning (2 mentions)
Dmem medium, by Corning (2 mentions)
Nu serum, by Corning (2 mentions)
28 protocols using glutamax
1
Cell Line Maintenance Protocol
2
Cell Viability Screening for Prostate Cancer
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Cells were seeded at the following densities: DU-145 (500 cells/0.1 mL), AD1 (2,000 cells/0.1 mL), R1D567 (1,000 cells/0.1 mL), LNCaP95 (4,000 cells/0.1 mL), 22Rv1 (2,000 cells/0.1 mL) in 96 well plates in RPMI media (Sigma-Aldrich) with 10% FBS, 1% penicillin-streptomycin, and 1% Glutamax (Corning). After an overnight incubation at 37°C and 5% CO2, media in the wells was replaced with fresh RPMI media with 5% charcoal-stripped (CSS)-FBS, 1% penicillin-streptomycin, and 1% Glutamax (Corning). Cells were then grown in the media for 3 days. One of the following three drug groups is then administered: enzalutamide, saracatinib, ranging from 0.39-100μM, and docetaxel 0.04-10nM. All drugs were obtained from Selleckchem. Treatment lasted for 6 days with replenishment of the media and drug after 3 days. Cell viability was measured using WST-1 at 1:10 dilution with CSS-FBS media at absorbance of 450 nm (Tecan 1100 Plate Reader). IC50 dosage was calculated using GraphPad Prism. Each data point was conducted in technical and biological triplicate.
3
MCF-7 and T47D Cell Culture Protocol
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MCF-7 and T47D cells were cultured in growth medium containing Dulbecco's minimum essential medium (DMEM) (Cellgro by Mediatech Inc, Manassas, VA) plus 10% fetal bovine serum (FBS) (Hyclone by Thermo Scientific, South Logan, UT), and 1% each of nonessential amino acids (NEAA) (Cellgro), Glutamax and Penicillin-streptomycin-neomycin (PSN) antibiotics mixture (Gibco by Invitrogen Corp. Carlsbad, CA) and maintained at 37°C and 5% CO2. For each experiment, MCF-7 cells are seeded in growth medium for 24 hr. The medium was then replaced with steroid-free medium containing phenol red-free DMEM plus 10% charcoal/dextran-stripped (cs) FBS, and 1% each of NEAA, Glutamax and PSN, and the cells were incubated at 37°C for 48–72 hr before treatment. The cells were pre-treated with 1 μM ICI 182,780 (ICI) or 1 μM in solution AMPK inhibitor compound C/Dorsomorphin (DOS) (Calbiochem, EMD Millipore Corp. Billerica, MA). The cells were treated simultaneously with the following, unless otherwise indicated: 10 ng/ml human tumor necrosis factor alpha (TNFα; Invitrogen), and 10 nM E2, 10 μM resveratrol (RES), 25 μM Rolipram (ROL), or 10 μM Forskolin (FSK) (Sigma–Aldrich Inc., St. Louis, MO).
4
Culturing HeLa, HEK293T, and mESCs
5
Generation and Transduction of NALM6 Cells
6
Differentiation of iPSCs to Macrophages
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See Supplemental Experimental Procedures for details of cells and assays used. iPSCs were differentiated to macrophages as previously described (van Wilgenburg et al., 2013 (link)). In short, 3 × 106 iPSCs were seeded into an Aggrewell 800 well (STEMCELL Technologies) to form EBs, in mTeSR1 and fed daily with medium plus 50 ng/mL BMP4 (Peprotech), 50 ng/mL VEGF (Peprotech), and 20 ng/mL SCF (Miltenyi Biotec). Four-day EBs were then differentiated in either 6-well plates (15 EBs/well), T75 (75 EBs), or T175 flasks (150 EBs) in X-VIVO15 (Lonza), supplemented with 100 ng/mL M-CSF (Invitrogen), 25 ng/mL IL-3 (R&D), 2 mM Glutamax (Invitrogen), 100 U/mL penicillin and 100 μg/mL streptomycin (Invitrogen), and 0.055 mM β-mercaptoethanol (Invitrogen), with fresh medium added weekly. pMacpre emerging into the supernatant after approximately 1 month were collected weekly and differentiation cultures replenished with fresh medium. Harvested cells were strained (40 μm, Corning) and used: either directly as pMacpre; or plated onto tissue-culture treated plastic or glass coverslips at 100,000 per cm2 and differentiated for 7 days or more to pMac in X-VIVO15 with 100 ng/mL M-CSF, 2 mM Glutamax, 100 U/mL penicillin, and 100 μg/mL streptomycin; or co-cultured with iPSC-derived neurons.
7
Human MEC Oncosphere Culture
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Human MEC cells were seeded at 500 cells per well in 24-well ultralow-attachment plates (Corning) and cultured in serum-free DMEM/F12 medium supplemented with penicillin-streptomycin (1%), GlutaMAX (2 nM), human EGF (20 ng/mL), human FGF2 (20 ng/mL), N-2 supplement (1%) and insulin (10 µg/mL). All the growth factors were ordered from Sigma-Aldrich and other supplements were ordered from ThermoFisher. After 2-week culture, spheres were photographed under microscope (Leica). Two biological repeats (1000 initial cells in total) were set up and used for analysis. The oncosphere size was presented based on the diameter measured by ImageJ (version 1.51J8).
8
Cell Line Cultivation Protocols for Cancer Research
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HEK293 cells, metastatic bladder cancer cell lines (TCC-SUP and 5637) and Natural Killer (NK)-92 cells were from DSMZ (Germany). Metastatic melanoma cell line (MelC), originating from human metastatic melanoma specimens, was obtained from the Department of Therapeutic Research and Medicines Evaluation, Pharmacogenetics (ISS Rome, Italy). Metastatic colon cancer cell line (HT29) was obtained from Mario Negri Institute of Milan (Italy). Human breast cancer (MDA-MB-231) cell line was from ATCC. HEK 293 cells were cultured in DMEM medium supplemented with 10% of FBS (Sigma), 1mM L-glutamine (Carlo Erba), glucose and 1mM non-essential amino acids (Gibco). MelC and 5637 cells were cultured in RPMI 640 medium with 10% of FBS and 1mM L-glutamine. TCC-SUP cells were grown in DMEM medium with 20% of FBS and 1mM L-glutamine. NK-92 cells were cultured in α MEM medium with 10% FBS and 2mM L-glutamine and IL-2 (75 IU/ml, R&D system). HT29 cells were grown in McCoy’s medium with 10% FBS and 1mM L-glutamine. MDA-MB-231 cells were cultured in DMEM medium (Corning) with 10% of FBS (Sigma) and 1X GlutaMAX (Corning). All cells were grown at 37°C in a 5% CO2 humidified incubator.
9
Glioma and Adrenal Cell Line Protocols
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The human glioma cell line MGM-1 was a gift from Dr Hiroaki Kataoka (University of Miyazaki). MGM-1 cells were grown in Dulbecco’s modified Eagle medium (Gibco, Thermo Fisher Scientific #11965092) with 10% heat-inactivated fetal bovine serum (Sigma-Aldrich #12306C) plus 100 IU/ml penicillin and 100 μg/ml streptomycin (Gibco, Thermo Fisher Scientific #15140122) at 37 °C and 5% CO2. The adrenal cortical carcinoma cell line NCI-H295R (referred to as H295R-S1; #CRL-2128) was purchased from the American Type Culture Collection and grown in Dulbecco’s modified Eagle medium/F-12, GlutaMAX with 2.5% Nu-Serum (Corning, #355100), 100 IU/ml penicillin, 100 μg/ml streptomycin, plus ITS+ Premix Universal Culture Supplement at a concentration recommended by the manufacturer (Corning, #354352). Cells were passaged using trypsin/EDTA 0.25% (Gibco, Thermo Fisher Scientific #25200056), and a maximum of 10 passages were used for all cell lines. For collection of cell pellets for CYP450 screening and antibody testing, MGM-1, MGM-3, NHA, HMC3, MA-10, and Huh7 cells were cultured and pelleted according to our previously published methods (22 , 66 ).
10
Culturing Human Breast Cancer Cell Lines
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JIMT-1 (AddexBio), BT-474 (ATCC), and SKBR-3 (ATCC) were cultured in RPMI1640 (Corning) supplemented with 10% EquaFETAL® (Atlas Biologicals), GlutaMAX® (2 mM, Gibco), sodium pyruvate (1 mM, Corning), and penicillin-streptomycin (penicillin: 100 units mL–1; streptomycin: 100 µg mL–1, Gibco). KPL-4 (provided by Dr. Junichi Kurebayashi at Kawasaki Medical School)51 (link) and MDA-MB-231 (ATCC) were cultured in DMEM (Corning) supplemented with 10% EquaFETAL®, GlutaMAX®(2 mM), and penicillin-streptomycin (penicillin: 100 units mL–1; streptomycin: 100 µg mL–1). All cells were cultured at 37 °C under 5% CO2 and passaged before becoming fully confluent up to 10 passages. All cell lines were periodically tested for mycoplasma contamination. Cells were validated for the HER2 expression level in cell-based ELISA prior to use (see the following Cell-based ELISA section).
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