HEK 293TT cells, purchased from the National Cancer Institute’s Developmental Therapeutics Program (Frederick, Maryland, USA), were grown in complete DMEM (ThermoFisher) containing 10% FBS (ThermoFisher), 100 U/mL penicillin, 100 μg/mL streptomycin (Dutscher), 1x Glutamax-I (ThermoFisher) and 250 μg/mL Hygromycin B (Sigma-Aldrich). RS cells (Evercyte, Vienna, Austria) were grown in Optipro (ThermoFisher) containing 100 U/mL penicillin, 100 μg/mL streptomycin, 1x Glutamax-I. GM95 cells purchased from the RIKEN BRC cell bank were cultured in DMEM with 10% FBS, 100 U/mL penicillin, 100 μg/mL streptomycin, and 1x Glutamax-I, and LNCaP cells were grown in RPMI medium supplemented with 10% of FBS, 100 U/mL penicillin, 100 μg/mL streptomycin, and 1x Glutamax-I. Cells were maintained at 37°C in a humidified 5% CO2 incubator, and passaged at confluence by trypsinization for 10 min with 1x TrypLE Express (ThermoFisher).
Glutamax 1
Manufactured by Thermo Fisher Scientific
Sourced in United States, United Kingdom, Germany, Japan, France, Belgium, Canada, Italy, Australia, China, Austria, Switzerland, Spain, Argentina, Netherlands
GlutaMAX-I is a cell culture supplement manufactured by Thermo Fisher Scientific. It serves as a stable and efficient alternative to L-glutamine for cell culture applications.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (195 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (158 mentions)
Streptomycin, by Thermo Fisher Scientific (107 mentions)
Penicillin, by Thermo Fisher Scientific (107 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (84 mentions)
Neurobasal medium, by Thermo Fisher Scientific (75 mentions)
B27 supplement, by Thermo Fisher Scientific (64 mentions)
Non essential amino acids (neaa), by Thermo Fisher Scientific (56 mentions)
Sodium pyruvate, by Thermo Fisher Scientific (37 mentions)
Hepes, by Thermo Fisher Scientific (36 mentions)
Fetal bovine serum (fbs), by Merck Group (36 mentions)
Rpmi 1640, by Thermo Fisher Scientific (34 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (28 mentions)
Dmem f12, by Thermo Fisher Scientific (27 mentions)
Poly d lysine, by Merck Group (26 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (26 mentions)
Trypsin edta, by Thermo Fisher Scientific (26 mentions)
2 mercaptoethanol, by Thermo Fisher Scientific (22 mentions)
Gentamicin, by Thermo Fisher Scientific (20 mentions)
Neurobasal a, by Thermo Fisher Scientific (19 mentions)
877 protocols using glutamax 1
1
Cell Culture Conditions for Various Cell Lines
2
Inflammation Evaluation in Cell Culture
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RPMI 1640 supplemented with 10% FBS (Sigma–Aldrich, Lot 111M3397)
Media tested for inflammation:
Media tested for inflammation:
3
Characterization of Prostate Cancer Cell Lines
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The human prostate cancer cell lines PC-3, DU145, 22Rv1, VCaP, and LNCaP were obtained from ATCC (Manassas, VA, USA). In literature PC-3 cells are described to be docetaxel-resistant [29 (link)]. All cell lines except LNCaP cells are androgen-independent and abiraterone/enzalutamide-resistant due to the absence of AR (PC-3 and DU145) or the presence of AR-V7 (22Rv1 and VCaP). Cells were incubated at 37°C in a humidified atmosphere with 5% (v/v) CO2. Cells were continuously kept in culture for a maximum of 3 months, and were routinely inspected microscopically for stable phenotype and regularly checked for contamination with mycoplasma. All cell lines were recently authenticated by a commercial service (Multiplexion, Heidelberg, Germany) using single nucleotide polymorphism (SNP)-profiling method.
PC-3, DU145 and 22Rv1 cells were cultured in 10% FBS/RPMI medium (RPMI medium supplemented with GlutamaxTM-I (Invitrogen, Paisley, UK) containing 10% fetal bovine serum (FBS, Invitrogen) and 1% penicillin/streptomycin (Invitrogen)). LNCaP cells were cultured in 10% FBS/RPMI medium (RPMI medium supplemented with GlutamaxTM-I containing 10% FBS, 1% penicillin/streptomycin, and 1 mM sodium pyruvate (Invitrogen)). VCaP cells were cultured in 10% FBS/DMEM medium (DMEM medium supplemented with GlutamaxTM-I (Invitrogen) containing 10% FBS and 1% penicillin/streptomycin (Invitrogen)).
PC-3, DU145 and 22Rv1 cells were cultured in 10% FBS/RPMI medium (RPMI medium supplemented with GlutamaxTM-I (Invitrogen, Paisley, UK) containing 10% fetal bovine serum (FBS, Invitrogen) and 1% penicillin/streptomycin (Invitrogen)). LNCaP cells were cultured in 10% FBS/RPMI medium (RPMI medium supplemented with GlutamaxTM-I containing 10% FBS, 1% penicillin/streptomycin, and 1 mM sodium pyruvate (Invitrogen)). VCaP cells were cultured in 10% FBS/DMEM medium (DMEM medium supplemented with GlutamaxTM-I (Invitrogen) containing 10% FBS and 1% penicillin/streptomycin (Invitrogen)).
4
Validation of GPC3+ Cancer Cell Lines
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G401 (rhabdoid tumor, ATCC) and HUH7 (hepatocellular carcinoma, a gift from Dr. Xiao-Tong Song, Baylor College of Medicine) were used as GPC3+ targets. The identity of HUH7 was confirmed by the Characterized Cell Line Core Facility at MD Anderson Cancer Center. A549 (lung carcinoma, ATCC) cells were used as negative controls. 293T cells (ATCC) were used for packaging viral vectors. HUH7, A549, and 293T cell lines were grown in DMEM (Thermo Scientific), G401 in alpha-MEM (Thermo Scientific) media supplemented with 10%–20% fetal bovine serum (FBS) (Thermo Scientific), and 2 mmol/L GlutaMAX-I (Invitrogen). The generation of eGFP.ffLuc-expressing HUH7, G401 and A549 cells has been previously described.8 (link) Human MSCs from healthy donors were obtained under a Baylor College of Medicine institutional review board (IRB)-approved protocol after informed consent was obtained in accordance to the Declaration of Helsinki. MSCs were cultured in αMEM (Lonza) supplemented with 20% FBS (Thermo Scientific) and 2 mmol/L GlutaMAX-I (Invitrogen; complete αMEM).
5
Cultivation of Procyclic and Bloodstream T. brucei
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Procyclic form T. brucei 427 strain containing T7 RNA polymerase and Tet repressor protein genes, respectively under control of G418 and hygromycin (clone 29.13.6 cells, kindly provided by George Cross) were grown at 28°C without CO2 in original SDM-79 medium [82 (link)] containing hygromycin B (Roche) at 50 μg/ mL, G418 (Invitrogen) at 15 μg/ mL, 15% (v/v) heat inactivated fetal bovine serum (FBS), 2 g/L sodium bicarbonate, fresh 2 mM Glutamax I (Invitrogen) and 22.5 mg / ml haemin (added from a 0.05 M stock in NaOH), adjusted to pH 7.3. Bloodstream form T. brucei 427 strain containing T7 RNA polymerase and Tet repressor protein, under control of G418, known as single markers cells, kindly provided by George Cross, were grown at 37°C with 5% CO2 in HMI-9T medium [83 (link)] containing fresh 2mM Glutamax I (Invitrogen) and 2.5 μg/ mL G418 (Invitrogen).
6
Circadian Regulation of Mouse Skin Fibroblasts
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Bmal1+/+ and Bmal1−/− adult mouse skin fibroblasts (MSF) cells were grown in Dulbecco’s Modified Eagle Medium (DMEM) containing 10% (v/v) HyClone III Serum (Analab; Cat # SH30109.03), 1/100 Glutamax-I (Invitrogen; Cat # 35050-038), 1/100 Penicillin-Streptomycin (SIGMA; Cat # P4333) and 1/500 MycoZap™ (Lonza; Cat # VZA-2022) in multiple six-well plates until fully confluent (n = 3, per time-point, per genotype). Confluent MSF cells were treated with 100 nM (final concentration) of dexamethasone (DEX) for 15 min to synchronize the cells. MSF cells were then washed three times with PBS (37 °C) and were incubated in HEPES-buffered Medium; 1× DMEM powder (SIGMA; Cat # D5030), 5 mg/ml 1, 2-13C2 (99%) D-glucose (Cambridge Isotope Laboratories, Cat # CLM-504-1), 0.35 mg/mL sodium bicarbonate, 0.01 M HEPES, 5% (v/v) HyClone III Serum, 1/100 Glutamax-I, 1× B-27 supplement (LifeTech Cat # 17504-044), 1× Non-Essential Amino acids, and 1/500 MycoZap™, pH 7.4 (adjusted with HCl) and osmolality 350 mOsm (adjusted with NaCl) at 37 °C under DD cycle. Twenty-four hours after the DEX treatment, MSF cells were harvested at every three-hour interval for two days for subsequent metabolic flux analysis.
7
Optimizing Cell Culture Conditions for HeLa and U2OS
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HeLa and U2OS cells (UCSF tissue culture facility) were cultured in Advanced DMEM (Invitrogen, Grand Island, NY) supplemented with 2% FBS (Invitrogen, Grand Island, NY) and Glutamax-I (Invitrogen, Grand Island, NY). Lyophilized roscovitine (Millipore, Billerica, MA) was resuspended in DMSO. HeLa cells were treated with 17 μM of nocodazole for 45 min to depolymerize microtubules. To inhibit CDK2 function, U2OS cells were incubated with 2 μM of roscovitine for 24 hr. Cells were transfected with siRNA using Oligofectamine (Invitrogen, Grand Island, NY) according to the manufacturer's instructions and analyzed 48 hr later. Sequences for the siRNA oligonucleotides used in this study are described in Supplementary file 2 . To deplete CEP215 and CEP63 using previously published sequences, HeLa cells were grown in DMEM (Invitrogen, Grand Island, NY) supplemented with 10% FBS, and Glutamax-I.
8
Prostate Cancer Cell Line Culturing
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Culture and handling of the cell lines was performed as described discribed previously20 . Human prostate cancer cell lines 22Rv1 (androgen-independent, AR-V7(+)) and LNCaP (androgen-dependent, AR-V7(−)), as well as human fibroblast cell line MRC-9 were used. 22Rv1 and LNCaP cells were purchased from ATCC (Manassas, VA, USA). MRC-9 cells were kindly donated by Prof. Dr. Dr. med. Sonja Loges (University Medical Center Hamburg-Eppendorf, Hamburg, Germany). For 22Rv1 and LNCaP cells 10% FBS/RPMI medium was used (RPMI medium supplemented with Glutamax-I (Invitrogen, Paisley, UK) containing 1% penicillin/streptomycin (Invitrogen) and 10% fetal bovine serum (FBS) (Invitrogen)). For MRC-9 cells 10% FBS/DMEM medium was used (DMEM medium supplemented with Glutamax-I (Invitrogen) containing 1% penicillin/streptomycin (Invitrogen) and 10% FBS (Invitrogen)). The cell lines were recently authenticated by a commercial service (Multiplexion, Heidelberg, Germany).
9
Culturing Trypanosoma brucei Cell Lines
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PCF
Trypanosoma brucei clone 29.13.6 cells, kindly provided by Prof. George Cross, were maintained in original SDM-79 (Invitrogen, Trypanosome Community, UK) containing 10% heat-inactivated fetal bovine serum (Gibco, Invitrogen), 2 g/L sodium bicarbonate, 2 mM Glutamax I (Invitrogen, UK), 7.5 mg/L haemin, 15 μg/ml G418 and 50 μg/ml hygromycin at 28°C without CO
2 (non-treated plastic culture flask, Thermo Scientific). Culture adapted strain 427 monomorphic BSF
T. brucei (variant 221, MITat 1.2) genetically modified to express T7 RNA polymerase and tetracycline repressor protein, known as Single Marker cells
10 (link), were cultured in HMI-9T medium, a modification of the original HMI-9 which contains 56 μM 1-thioglycerol instead of 200 μM 2-mercaptoethanol, 10% heat-inactivated fetal bovine serum (Gibco, Invitrogen), 2 mM Glutamax I, 2.5 μg/mL G418 at 37°C with 5% CO
2.
Trypanosoma brucei clone 29.13.6 cells, kindly provided by Prof. George Cross, were maintained in original SDM-79 (Invitrogen, Trypanosome Community, UK) containing 10% heat-inactivated fetal bovine serum (Gibco, Invitrogen), 2 g/L sodium bicarbonate, 2 mM Glutamax I (Invitrogen, UK), 7.5 mg/L haemin, 15 μg/ml G418 and 50 μg/ml hygromycin at 28°C without CO
2 (non-treated plastic culture flask, Thermo Scientific). Culture adapted strain 427 monomorphic BSF
T. brucei (variant 221, MITat 1.2) genetically modified to express T7 RNA polymerase and tetracycline repressor protein, known as Single Marker cells
10 (link), were cultured in HMI-9T medium, a modification of the original HMI-9 which contains 56 μM 1-thioglycerol instead of 200 μM 2-mercaptoethanol, 10% heat-inactivated fetal bovine serum (Gibco, Invitrogen), 2 mM Glutamax I, 2.5 μg/mL G418 at 37°C with 5% CO
2.
10
Neuronal Gene Expression Profiling
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The hippocampus and brainstem were isolated from E16.5 and E12.5–E14.5 embryos, respectively, and digested with papain (Worthington Biochemical Corporation). The dissociated cells were nucleofected with the γA3-Venus (or -tdTomato) or γB2-tdTomato plasmid using the P3 Primary Cell Electroporation Kit (Lonza, Germany). The cells were resuspended, plated on 1 mg/ml poly-L-lysine–coated glass coverslips, and cultured in Minimum Essential Medium (Invitrogen) containing 5% fetal bovine serum, B27 supplement (Invitrogen), and GlutaMax I (Invitrogen). After 48 h, the medium was replaced with Neurobasal serum-free medium (Invitrogen) supplemented with B27 and GlutaMax I. Neurons were fixed with 4% paraformaldehyde or methanol for 15 min and processed for immunocytochemistry.
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