129/Ola-derived E14tg2a ESCs (provided by H. Niwa, RIKEN) and C57BL/6N-derived RENKA ESCs (provided by K. Sakimura, Niigata University) were maintained on mitomycin C-treated mouse embryonic fibroblast (MtCMEF) cells in 2i medium composed of KnockOut DMEM (Thermo Fisher Scientific), 15% KnockOut Serum Replacement (Thermo Fisher Scientific), 2 mM l -alanyl-l -glutamine solution (Wako), 5 μg/ml insulin (Sigma), 1 μM PD0325901 (Wako), 3 μM CHIR99021 (Chemscene), mouse LIF (prepared in-house with a final concentration higher than 1000 U/ml), 1 mM sodium pyruvate (Sigma), 0.1 mM non-essential amino acids (Sigma), 0.1 mM 2-mercaptoethanol (Sigma), and 50 U/ml penicillin/streptomycin (PS) (Sigma) (31 (link)). When E14tg2a-derived cells were used for experiments, the cells were cultured on gelatin-coated plates with LIF medium composed of DMEM (Thermo Fisher Scientific), containing 10% fetal bovine serum (FBS) (Biosera), mouse LIF, 1 mM Na pyruvate, 0.1 mM non-essential amino acids, 0.1 mM 2-mercaptoethanol and PS. All other cells were cultured in DMEM supplemented with 10% FBS and PS. The recombinant lentiviruses were prepared as previously described (32 (link)).
L alanyl l glutamine solution
Manufactured by Fujifilm
Sourced in Japan
L-alanyl-L-glutamine solution is a cell culture supplement that provides a stable source of the amino acids L-alanine and L-glutamine. It is designed to support cell growth and metabolism in in vitro cell culture applications.
Automatically generated - may contain errors
Lab products found in correlation
Gentamicin, by Fujifilm (3 mentions)
Non essential amino acids (neaa), by Fujifilm (2 mentions)
Stemsure d mem, by Fujifilm (2 mentions)
Knockout dmem, by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin p s, by Merck Group (1 mentions)
Sodium pyruvate, by Merck Group (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
Knockout serum replacement (ksr), by Thermo Fisher Scientific (1 mentions)
Non essential amino acids (neaa), by Merck Group (1 mentions)
Insulin, by Merck Group (1 mentions)
2 mercaptoethanol, by Merck Group (1 mentions)
Fetal bovine serum (fbs), by Biosera (1 mentions)
Pd0325901, by Fujifilm (1 mentions)
Non essential amino acids (neaa), by Biosera (1 mentions)
Chir99021, by ChemScene (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by GE Healthcare (1 mentions)
Dulbecco modified eagle medium (dmem), by Fujifilm (1 mentions)
Monothioglycerol solution, by Fujifilm (1 mentions)
Pd0325901, by ChemScene (1 mentions)
6 protocols using l alanyl l glutamine solution
1
Maintenance and Culture of Mouse ESCs
2
Culturing Mouse Embryonic Stem Cells
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Wild-type (WT) mESCs derived from inbred mice (Bruce 4 C57BL/6 J, male, EMD Millipore, Billerica, MA, USA) and other knock-in derivatives were cultured as described previously32 (link). C57BL/6NCr (male) mESCs17 (link) were used as Sox2 STREAMING-tag knock-in cells. Briefly, all mESC lines were maintained in 2i medium (Dulbecco’s modified Eagle’s medium [DMEM, Wako, Osaka, Japan, 197-16275]; 15% fetal bovine serum [FBS, GE Healthcare, Little Chalfont, UK, SH30396.03]); 0.5 mM monothioglycerol solution [Wako, 195-15791]; 1×MEM nonessential amino acids [Wako, 139-15651]; 2 mM L-alanyl-L-glutamine solution [Wako, 016-21841]; 1,000 U/mL leukemia inhibitory factor [Wako, 195-16053]; 20 µg/mL gentamicin [Wako, 078-06061]; 3 µM CHIR99021 [Cayman Chemical, Ann Arbor, MI, USA, 13122]; and 1 µM PD0325901 [Chemscene, Monmouth Junction, NJ, USA, CS-0062]) on a 0.1% gelatin (Sigma-Aldrich, St. Louis, MO, USA, G1890-100G)-coated dish at 37 °C and 5% CO2. The cell lines used in this study are listed in Supplementary Data 1 .
HeLa cells (CCL-2, ATCC) were grown in DMEM high-glucose medium (Nacalai Tesque, Kyoto, Japan) containing 10% FBS (Gibco, Grand Island, NY, USA) and 1% L-glutamine–penicillin–streptomycin solution (GPS; Sigma-Aldrich, St. Louis, MO, USA) at 37 °C in a 5% CO2 atmosphere.
HeLa cells (CCL-2, ATCC) were grown in DMEM high-glucose medium (Nacalai Tesque, Kyoto, Japan) containing 10% FBS (Gibco, Grand Island, NY, USA) and 1% L-glutamine–penicillin–streptomycin solution (GPS; Sigma-Aldrich, St. Louis, MO, USA) at 37 °C in a 5% CO2 atmosphere.
3
Culturing C57BL/6 mESCs in 2i Conditions
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Bruce 4 C57BL/6 mESCs (Merk Millipore, Billerica, MA) (and later derivatives) were cultured in 2i conditions (StemSure D-MEM [Wako Pure Chemicals, Osaka, Japan], 15% fetal bovine serum, 0.1 mM β-mercaptoethanol, 1 × MEM nonessential amino acids [Wako Pure Chemicals], 2 mM l -alanyl-l -glutamine solution [Wako Pure Chemicals], 1000 U/mL LIF [Wako Pure Chemicals], 20 µg/mL gentamicin [Wako Pure Chemicals], 3 µM CHIR99021, and 1 µM PD0325901) on a 0.1% gelatin-coated dish. Before each experiment, cells were passaged two times and cultured in 2i conditions as well as serum conditions (StemSure D-MEM, 15% fetal bovine serum, 0.1 mM β-mercaptoethanol, 1 × MEM nonessential amino acids, 2 mM l -alanyl-l -glutamine solution, 1000 U/mL LIF, 20 µg/mL gentamicin), or in serum conditions containing 2i inhibitors or PD0325901 at several concentrations, as described in Supplementary Table S1 . TSA and 5-AzaC were added to the cells at a final concentration of 50 nM and 50 μM, respectively. They were applied for 72 hours.
4
2i Culture of Mouse Embryonic Stem Cells
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WT mESCs (Bruce 4 C57BL/6J, male, EMD Millipore, Billerica, MA) and later derivatives were cultured under 2i conditions (StemSure D-MEM [Wako Pure Chemicals, Osaka, Japan], 15% of fetal bovine serum, 0.1 mM β-mercaptoethanol, 1 × MEM nonessential amino acids [Wako Pure Chemicals], a 2 mM L-alanyl-L-glutamine solution [Wako Pure Chemicals], 1000 U/mL LIF [Wako Pure Chemicals], 20 mg/mL gentamicin [Wako Pure Chemicals], 3 mM CHIR99021, and 1 mM PD0325901) in a 0.1% gelatin-coated dish.
5
Hypoxia-Induced GAG Expression in HBMEC Cells
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HBMEC/ciβ was cultured as described previously (15 (link)). Briefly, HBMEC/ciβ (1.7 × 106 cells) was cultured in VascuLife Basal Medium (Lifeline Cell Technology, Frederick, MD) supplemented with 10% FBS, 2 mm l -Alanyl-l -Glutamine Solution (FUJIFILM Wako Pure Chemical Co., Tokyo, Japan), 4 μg/ml blasticidin S (InvivoGen, San Diego, CA), 1% penicillin, and 1% streptomycin in an atmosphere of 5% CO2/95% air at 33 °C. HBMEC/ciβ (1.0 × 107 cells) were exposed to 24 h of hypoxia (pO2 < 1%) using the BIONIX-1 hypoxic cell culture kit (Sugiyamagen, Tokyo, Japan) and 30-min reoxygeneration to examine the effect of hypoxia/reperfusion on the expression level of GAGs (49 (link)). HEK293 cells were cultured in DMEM supplemented with 10% FBS, 100 units/ml penicillin G, and 50 units/ml streptomycin in an atmosphere of 5% CO2/95% air at 37 °C.
6
Generating NUGC4 and MKN45 Cell Lines for Galectin-4 Research
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Human gastric cancer NUGC4 and MKN45 cells were obtained from RIKEN BioResource Center (Tsukuba, Japan). Control cells (Con), which were transfected with non-coding crRNA instead of galectin-4 specific crRNA in CRISPR/Cas9-mediated genome-editing and rescue cells (Res), galectin-4 re-expressing cells that were established by transferring the galectin-4-expressing plasmid into a KO clone were established as described previously [15 (link)]. Cells were cultured in RPMI 1640 medium (FUJIFILM Wako Pure Chemical, Osaka, Japan) supplemented with 2 mM L-alanyl-L-glutamine solution (FUJIFILM Wako Pure Chemical) and 10% fetal calf serum (FCS) (Funakoshi Co., Ltd., Tokyo, Japan). Cells were washed with phosphate-buffered saline to completely remove FCS and collected by centrifugation at 500× g for 5 min to yield cell pellets.
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