253G1 line (passage number: P32 – P38), 454E2 line (P34), and RIKEN-2A line (P19) (RIKEN Cell Bank, Ibaraki, Japan.) were used in this study. All experiments were performed in accordance with ethical guidelines of our university and the cell bank. Undifferentiated hiPS were maintained on SNL 76/7 cells (ECACC, Salisbury, UK) as a feeder layer, as previously described18 (link). The undifferentiated hiPS cells were cultured in Dulbecco’s modified Eagle medium/F12 (DMEM/F12, Sigma-Aldrich, St. Louis, MO, USA) supplemented with 20% (v/v) knockout serum replacement (KSR, Life Technologies, Carlsbad, CA, USA), 0.1 mM nonessential amino acid (NEAA, Life Technologies), 2 mM L-glutamine (Sigma-Aldrich), 0.1 mM 2-mercaptoethanol (Nacalai Tesque, Kyoto, Japan), and 5 ng/mL basic fibroblast growth factor (bFGF, Wako Pure Chemical Industries, Osaka, Japan) in a humidified atmosphere of 5% CO2 and 95% air at 37 °C. The hiPS cells were subcultured every three days using CTK solution (0.25% (v/v) trypsin and 0.1 mg/mL collagenase (Nitta Gelatin Inc., Osaka, Japan) in PBS (-) supplemented with 20% (v/v) KSR and 1-mM calcium chloride). The details of the automated hiPS culture are described in Fig. 2 . The manual culture was conducted by an expert with five years of experience with hiPS culture.
Non essential amino acid neaa
Manufactured by Thermo Fisher Scientific
Sourced in United States
Non-essential amino acids (NEAAs) are a group of amino acids that can be synthesized by the human body and are not required to be obtained from dietary sources. They serve as the building blocks for proteins and play a crucial role in various physiological processes.
Automatically generated - may contain errors
Lab products found in correlation
Trypsin edta, by Thermo Fisher Scientific (3 mentions)
Knockout serum replacement (ksr), by Thermo Fisher Scientific (2 mentions)
Foetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Sodium pyruvate, by Thermo Fisher Scientific (2 mentions)
L glutamine, by Thermo Fisher Scientific (2 mentions)
2 mercaptoethanol, by Nacalai Tesque (1 mentions)
L glutamine, by Merck Group (1 mentions)
Trypsin, by Nitta Gelatin (1 mentions)
Collagenase, by Nitta Gelatin (1 mentions)
Penicillin streptomycin pen strep, by Thermo Fisher Scientific (1 mentions)
In fusion cloning, by Takara Bio (1 mentions)
Fetal bovine serum (fbs), by GE Healthcare (1 mentions)
L glutamine, by GE Healthcare (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Rock inhibitor y 27632, by Merck Group (1 mentions)
Penicillin, by Corning (1 mentions)
Streptomycin, by Corning (1 mentions)
Mem vitamin solution, by Merck Group (1 mentions)
Basic fibroblast growth factor (bfgf), by Thermo Fisher Scientific (1 mentions)
Dl lactic acid, by Merck Group (1 mentions)
7 protocols using non essential amino acid neaa
1
Automated Pluripotent Stem Cell Culture
2
Caco-2 Cell Monolayer Differentiation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Caco-2 cells at passage 45 (Sigma-Aldrich, Stockholm, Sweden) were cultured in Dulbecco's Modified Eagle's Medium (DMEM) (Life Technologies Invitrogen AB, Lidingo, Sweden), with 10% Foetal Bovine Serum (FBS) (Life Technologies), 1% NonEssential Amino Acid (NEAA) (Life Technologies), 100 IU/mL Penicillin Streptomycin (Pen-Strep) (Life Technologies). The cells were incubated in 37 °C with 5% CO2 and cultured in a cell culture flask (BD Falcon®, VWR International, Stockholm, Sweden) and the medium was changed every other day. The cells were cultured for approximately 1 week before they were detached (0.25% trypsin-EDTA, Life Technologies) and seeded in 12 well plates with semipermeable filter inserts (3 μm pore size, BD Biosciences, Le Pont de Claix, France), 50,000 cells/well. At confluence the culture medium was changed to FBS-free in both the upper and lower well compartment and to the lower compartment also a mixture of 10 μL/mL insulin, 0.55 μg/mL transferring and 6.7 ng/mL selenium (ITS, Life Technologies) was added to establish cell polarity and structural differentiation. The cell monolayers were let to cultivate for 14 days before experimentation. After 14 days, some cells were fixed in Karnowsky's fixative overnight as previously describe (Helander and Fändriks, 2014 (link)) for morphological analysis.
3
Plasmid Construction and Cell Line Maintenance
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Plasmids were constructed by standard molecular biology methods such as polymerase chain reaction (PCR) and In-fusion cloning (Clontech). Mutations for specific amino acids were generated by overlap-extension PCR. All cloning junctions and PCR products were verified by sequencing process. The pcDNA3 vector was used for the expression in mammalian cells, and pLL3.7 vector was used for virus production. Full plasmid sequences are available upon request, and main constructs will be available in Addgene after publication.
Cell culture and reagents HEK293A cells were maintained in Dulbecco's modified Eagle medium (DMEM) supplemented with 2 mM L-glutamine (GE Healthcare Life Sciences), 10% fetal bovine serum (FBS, GE Healthcare Life Sciences), 1 unit/mL penicillin (Corning), 100 g/mL streptomycin (Corning), non-essential amino acid (NEAA, Life Technologies), and 1 mM sodium pyruvate (Life Technologies). U87-MG cells were maintained in Dulbecco's modified Eagle medium (DMEM) supplemented with 2 mM L-glutamine, 10% FBS, 1 unit/mL penicillin, 100 g/mL streptomycin, and 1 mM sodium pyruvate. Cells were cultured in a humidified 95% air, 5% CO2 incubator at 37°C. For the transient transfection, we used Lipofectamine 2000 (Life Technologies) or lentiviruses which were prepared by KIST Virus Facility. Src inhibitor PP2, ROCK inhibitor Y-27632 were purchased from Sigma.
Cell culture and reagents HEK293A cells were maintained in Dulbecco's modified Eagle medium (DMEM) supplemented with 2 mM L-glutamine (GE Healthcare Life Sciences), 10% fetal bovine serum (FBS, GE Healthcare Life Sciences), 1 unit/mL penicillin (Corning), 100 g/mL streptomycin (Corning), non-essential amino acid (NEAA, Life Technologies), and 1 mM sodium pyruvate (Life Technologies). U87-MG cells were maintained in Dulbecco's modified Eagle medium (DMEM) supplemented with 2 mM L-glutamine, 10% FBS, 1 unit/mL penicillin, 100 g/mL streptomycin, and 1 mM sodium pyruvate. Cells were cultured in a humidified 95% air, 5% CO2 incubator at 37°C. For the transient transfection, we used Lipofectamine 2000 (Life Technologies) or lentiviruses which were prepared by KIST Virus Facility. Src inhibitor PP2, ROCK inhibitor Y-27632 were purchased from Sigma.
4
GDNF-Supplemented Mouse Embryonic Stem Cell Culture
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
GDNF-mESCCM consisted of high-glucose DMEM supplemented with 15% (v/v) heat-inactivated FBS, 0.1 mM β-mercaptoethanol (Gibco Invitrogen, Grand Island, NY, USA), 1% (v/v) non-essential amino acid (NEAA; Gibco Invitrogen, USA), 2 mM l -glutamine (Gibco Invitrogen, USA), 1% (v/v) antibiotic–antimycotic solution, 1,000 U/mL mouse leukemia inhibitory factor (mLIF; Chemicon International, Inc., Temecula, CA, USA), and 10 ng/mL GDNF (R&D Systems, Inc., Minneapolis, MN, USA). MEM alpha medium (Gibco Invitrogen, USA), which served as pSSCCM, was supplemented with 1% (v/v) antibiotic–antimycotic solution, 1% (v/v) NEAA, 0.1 mM β-mercaptoethanol, N2-1 supplement (Merck Millipore; Darmstadt, Germany), DL-lactic acid (Sigma-Aldrich, USA), 1% (v/v) MEM vitamin solution (Sigma-Aldrich, USA), 30 ng/mL β-estradiol (Sigma-Aldrich, USA), 1% (v/v) heat-inactivated FBS, 10 ng/mL basic fibroblast growth factor (bFGF; PeproTech, Inc., Rocky Hill, NJ, USA), 20 ng/mL epidermal growth factor (EGF; PeproTech, Inc., USA), 1,000 U/mL mLIF, and 10 ng/mL GDNF.
5
Generating Fabry Disease iPSCs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Fabry disease (FD) fibroblasts were individually biopsied from four patients under the approval of the AMC Institutional Review Board (2011-0451). Studies using human materials including iPSCs were performed under the approval of the Institutional Review Board of KAIST (KH2016-52). Fibroblasts were cultured in DMEM (Welgene, Seoul, Korea) containing 10% fetal bovine serum (FBS, Invitrogen, Carlsbad, CA), 1% penicillin-streptomycin (Invitrogen) at 37°C, 5% CO2 in air. Retroviruses expressing OCT4, SOX2, cMYC, and KLF4 were transfected into FD fibroblasts to generate iPSCs as previously described [23] (link). Wild type (WT)-iPSCs derived from foreskin fibroblasts (CRL-2097, ATCC, Manassas, VA) were used as a control. iPSCs were cultured in human embryonic stem cell culture medium containing DMEM/F12 (Invitrogen, Carlsbad, CA), 20% KnockOut™ Serum Replacement (Invitrogen), supplemented with 10 ng/mL bFGF2 (R&D systems, Minneapolis, MN), 1% non-essential amino acid (NEAA) (Invitrogen), 1% penicillin and streptomycin (Invitrogen) on mitomycin C-treated MEF-seeded culture plates at 37°C, 5% CO2 in air. After detachment with dispase (Invitrogen), human iPSCs were passaged to fresh plates every 6 d. The GLA mutations were identified from genomic DNAs extracted from FD-iPSCs and fibroblasts by direct sequencing. The primers used for detecting the mutations are listed in Table S1.
6
Anticancer Drug Cytotoxicity Evaluation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
L-Canavanine (purity ≥98%), doxorubicin (≥97%), cisplatin (≥97%), and 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT; ≥98%) were purchased from Sigma-Aldrich GmbH, Munich, Germany. DMEM, RPMI 1640, non-essential amino acid (NEAA), sodium pyruvate, penicillin, streptomycin, foetal bovine serum (FBS), trypsin-EDTA, L-glutamine, and dimethylsulfoxide (DMSO) were purchased from Gibco® Invitrogen, Darmstadt, Germany.
7
Characterization of ABCB1 and ABCG2 in Gefitinib and Erlotinib Metabolism
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Gefitinib (purity 98%) and erlotinib (purity 98%) were purchased from Wuhan Sunrise Technology Development, Inc. (Wujiashan, China). O-Desmethyl gefitinib (purity 98%) was obtained from Toronto Research Chemicals Inc. (North York, ON, Canada). R123, dimethyl sulfoxide (DMSO), sodium dodecyl sulfate (SDS), quinidine (purity 98%), 3-(4′,5′-dimethylthiazol-2′-yl)-2,5-diphenyltetrazolium bromide (MTT), Triton X-100 and formic acid were provided by Sigma (St. Louis, MO, USA). Acetonitrile, ethyl acetate and methanol with LC-MS grade were obtained from J.T. Baker Inc. (Center Valley, PA, USA). Fetal bovine serum (FBS) was obtained from Biological Industries Inc. (Kibbutz, Beit Haemek, Israel). Dulbecco’s modified Eagle medium (DMEM), Hank’s buffered salt solution (HBSS), 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), nonessential amino acid (NEAA) and trypsin/EDTA were purchased from Invitrogen (Grand Island, NY, USA). Antibodies against ABCB1 and ABCG2 were supplied by Abcam (Cambridge, UK). Antibodies against CYP3A4, CYP2D6 and β-actin were purchased from GeneTex (San Antonio, TX, USA). Polyvinylidene fluoride transfer membranes (Immobilon P) and chemiluminescence (ECL) were obtained from Millipore Corp. (Millipore, Bedford, MA, USA). Milli-Q plus water (Millipore, Bedford, MA, USA) was used for all processes.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!