The human kidney-derived podocyte cell line PODO/TERT256 (Evercyte, Vienna, Austria, CHT-033-0256) was used in this study. We have signed MTAs with companies. PODO/TERT256 cells are known to maintain physiological properties of primary cells and can be cultured extensively [60 (link)]. The cells were cultured at 37 °C in a 5% CO2 environment using cell culture dishes pre-coated with human collagen I. The cells were fed with MCDB 131 medium (Thermo Fisher Scientific, Waltham, MA, USA) which has the same components as the recommended medium, supplemented with GlutaMAX-I (1.6 mM) (Thermo Fisher Scientific, Waltham, MA, USA), Bovine Brain Extract (9.6 μg/mL) (Lonza, Basel, Switzerland), hEGF (8 ng/mL) (Sigma-Aldrich, St. Louis, MO, USA), Hydrocortisone (20 ng/mL) (Sigma-Aldrich, St. Louis, MO, USA), G418 (100 µg/mL) (Fujifilm Wako, Osaka, Japan), and 20% fetal bovine serum. The number of podocytes was determined based on the specific requirements of each experiment. After 7–10 days of differentiation, cells were subjected to experimental treatment. Treatment with each drug was carried out under serum-free conditions.
Glutamax 1
Manufactured by Thermo Fisher Scientific
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About the product
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.
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Fetal bovine serum (fbs), by Thermo Fisher Scientific (340 mentions)
Streptomycin, by Thermo Fisher Scientific (244 mentions)
Penicillin, by Thermo Fisher Scientific (236 mentions)
B27 supplement, by Thermo Fisher Scientific (160 mentions)
Neurobasal medium, by Thermo Fisher Scientific (159 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (152 mentions)
Non essential amino acids (neaa), by Thermo Fisher Scientific (131 mentions)
Hepes, by Thermo Fisher Scientific (106 mentions)
Sodium pyruvate, by Thermo Fisher Scientific (88 mentions)
Dmem f12, by Thermo Fisher Scientific (82 mentions)
Fetal bovine serum (fbs), by Merck Group (79 mentions)
Rpmi 1640, by Thermo Fisher Scientific (78 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (75 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (56 mentions)
Penicillin streptomycin, by Merck Group (56 mentions)
Fetal calf serum (fcs), by Thermo Fisher Scientific (55 mentions)
N2 supplement, by Thermo Fisher Scientific (53 mentions)
Gentamicin, by Thermo Fisher Scientific (52 mentions)
Trypsin edta, by Thermo Fisher Scientific (49 mentions)
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Market Availability & Pricing
GlutaMAX™-I Supplement is an officially listed product from Thermo Fisher Scientific that is available through authorized distributors. The product typically retails for $61.65 to $68.50 per 100 mL bottle.
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Get pricing insights and sourcing optionsSpelling variants (same manufacturer)
GlutaMAX - 24361 citations
IMDM Glutamax I - 15 citations
L-alanyl-L-glutamine (Glutamax I; - 11 citations
αMEM + GlutaMAX-I - 9 citations
GlutaMAX-I Gibco - 8 citations
RPMI 1640 medium with Glutamax I - 7 citations
RPMI 1640 W/GLUTAMAX-I - 6 citations
Glutamax-I (1×) - 3 citations
DULBECCO'S MEM with Glutamax-1 - 3 citations
RPMI-1640- GlutaMAX-I without phenol red - 3 citations
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Product FAQ
2 281 protocols using «glutamax 1»
1
Cultivation of Immortalized Kidney Podocytes
2
Culturing UMPED83 Glioma Cells
All cell lines were maintained at 37°C with 5% CO2. Exclusion of Mycoplasma contamination was monitored and conducted by test with EZ-PCR kit (Biological Industries # 20-700-20). HEK293 cells were cultured in DMEM supplemented with 10% FBS and 1% P/S. UMPED83 (male, generated in the laboratory of Dr. Carl Koschmann from the University of Michigan) were cultured in Tumor Stem Media (TSM) consisting of a 1:1 mixture of DMEM/F12 (Invitrogen, 31330038) and Neurobasal -A (Invitrogen, 10888022) media, with 1:100 addition of HEPES Buffer Solution (Invitrogen 15630-080), MEM Sodium Pyruvate Solution (Invitrogen, 11360-070), MEM Non-Essential Amino Acids Solution (Invitrogen, 11140-050), GlutaMAX-I (Invitrogen, 35050-061) and Antibiotic-Antimycotic (Invitrogen,15240-096). The following additive were added freshly: B27 -A (1:50, Invitrogen, 12587010), human FGF (20 ng/mL, Shenandoah Biotechnology, 100–146), human EGF (20 ng/mL, Shenandoah Biotechnology, 100-26), human PDGF-AA (20 ng/mL, Shenandoah Biotechnology, 100-16), human PDGF-BB (20 ng/mL, 100-18, Shenandoah Biotechnology) and heparin (10 ng/mL, SIGMA, H3149). Adherent growth of these cells was acquired by coating wells with Laminin for 2h at 37°C (30 μg/ml, SIGMA, L2020). Cells were grown as monolayer for two passages before the experiments were conducted. Cell were plated in parallel for CellTitel-Glo in 96 well plates, and for H3-K27M-H3K27ac single-molecule analysis in 6 well plates. For single-molecule analysis media was removed at indicated time points, centrifuged at 21kg for 10min, aliquoted, flash-frozen and stored at −80 °C. Cell viability was assessed using CellTiter-Glo assay (#G7572, Promega, USA) in accordance with the manufacture protocol.
3
Isolation and Culture of Intestinal Organoids
Isolation of intestinal crypts and culture of small intestinal organoids were performed according to previously reported protocols with some modifications (Zhao et al. 2019 ). Briefly, anesthetized mice were dissected, and the mouse intestine was isolated, cut longitudinally, and washed twice with cold DPBS. The villi were gently scraped off with a sterile scalpel, after which the intestine was cut into small pieces (approximately 5 mm) and incubated with 10 mM EDTA (Gibco) in DPBS for 35 min at 4 °C. After removing EDTA, the small pieces were suspended by pipetting with cold DPBS using a 10 ml pipette. The crypt-rich supernatant was passed through a 70 μm cell strainer (BD Falcon) and centrifuged at 800 rpm for 5 min. Discard the supernatant and harvest the cell pellet after centrifugation, which is the desired crypt. The obtained crypts were embedded in Matrigel (Corning) and then seeded on 24-well plates. After Matrigel polymerization, ENR crypt culture medium (Advanced DMEM/F12 (Gibco), supplemented with GlutaMAX-I (Gibco), Penicillin/ Streptomycin (Gibco), G27 (bioGenous), N2 (Gibco), and N-acetylcysteine (Sigma-Aldrich), containing 500 ng/ml recombinant human R-spondin1 (bioGenous), 100 ng/ml recombinant mouse Noggin (bioGenous) and 50 ng/ml recombinant mouse EGF (bioGenous). Medium was changed every 2~3 days. For chemical treatment, 1 μM 4-Hydroxytamoxifen (4-OHT, Sigma-Aldrich, H6278), 2 μM Panobinostat (Selleckchem), and 2 μM Entinostat (Selleckchem) were added to the culture medium.
4
Inducible SRSF2 Expression in Lung Cancer Cells
Human LUAD cell lines (H358, H1299, and A549) were authenticated by DNA Short Tandem Repeat (STR) (ATCC Cell Line Authentification Service, LGC Standards, Molsheim, France) and routinely tested for mycoplasma contamination. Human retinal pigmental epithelial (RPE) (p53-/-) cell line was provided by Dr Sylvie Noordermeer (Leiden University Medical Center, LUMC, Oncode Institute, Leiden, The Netherlands). H358, H1299, and A549 cells were cultured in RPMI-1640 medium/L-GlutaMAX supplemented with 10% (v/v) fetal calf serum (FCS) as previously described [29 (link)]. RPE cells were maintained in Dulbecco’s modified Eagle’s medium GlutaMAX-I (Gibco) with 10% FCS. Generation of stable SRSF2-inducible clones was performed using a modified tetracyclin-regulated inducible expression system (Tet-On System, Clontech). Briefly, H358-Tet-On 4 cells, that stably express the Tet-repressor protein, were co-transfected with pTRE-SRSF2 and pTK-Hyg plasmids using Fugene 6 (Roche Diagnostic, France). Double transfectants cultured in 100 × 20-mm culture dishes were selected for 4 weeks in the presence of hygromycin B (200 μg/ml) and geneticin (G418; 800 μg/ml), and resistant clones were further isolated by successive sub-cultures in 96-, 24-, and 6-well plates. Clones were then screened for SRSF2 induction by western blotting following 24 h of 1-μg/ml doxycyclin treatment. Two clones were arbitrarily selected for further experiments. Other plasmids used in this study were pcDNA3.1-SRSF2-Hemagglutinin (HA) and pcDNA3.1-SRSF2(P95H)-HA encoding HA-tagged SRSF2 wild-type or SRSF2(P95H) mutant protein, pcDNA3.1-MRE11 (a kind gift from Dr MH Gatei, University of Queensland, Australia), pcDNA3.1-NBS1 (a kind gift from Dr JJ Mendiola, The Scripps Research Institute, La Jolla), pGEX-SRSF2(1–60), pGEX-SRSF2(60–115), and pGEX-SRSRF2(115–221) [30 (link)]. Transfection of plasmid DNA was performed using XtremeGENE 9 (Roche Diagnostics), Lipofectamine 3000 (Invitrogen), or JetPRIME (Polyplus Transfection, Illkirch, France), according to the manufacturer’s instructions. Cells were analyzed between 24 and 48 h after transfection. Cisplatin (cat. #PZ0383, calbiochem), hydroxurea (cat. #H8627), mirin (cat. #M9948), PFM01 (cat. #SML1735), 5,6-dichlorobenzimidazole 1-β-d -ribofuranoside (DRB; cat. #D1916), alpha-amanitin (cat. #A2263), palbociclib (cat. #PZ0383), PHA-767491 (cat. #PZ0178), and roscovitin (cat. #R7772) were from Sigma–Aldrich (Saint Quentin Fallavier, France).
5
Cell Culture Conditions for Various Cell Lines
HUVECs and BAECs were cultured in EGM-2 (Lonza, Basel, Switzerland). Cells with fewer than five passages were used in the experiments. Transgenic CHO cells were maintained in an F-12 Nutrient Mixture with GlutamaxTM-I (Thermo Fisher Scientific, MA, USA), 10% fetal bovine serum (FBS; Gibco, USA), and appropriate selection reagents, as described below. CHO-K1 cells were maintained in F-12 Nutrient Mixture with GlutamaxTM-I and 10% FBS. HAVSMCs were cultured in Dulbecco’s modified Eagle’s medium/F12 (DMEM/F12) (Nacalai Tesque, Japan) supplemented with 1% penicillin-streptomycin (Fujifilm, Japan) and 10% FBS. A10 cells were grown in DMEM (Wako, Osaka, Japan) supplemented with 10% FBS and 1% penicillin-streptomycin. NRK52E and NRK49F cells (ECACC, UK) were cultured in DMEM (Wako, Japan) supplemented with 5% FBS and the appropriate selection reagents. Gene transcription in CHO cells was induced by adding 100 ng/mL doxycycline (Merck KGaA, Darmstadt, Germany). Cells were incubated at 37℃ in 5% CO2 and 95% air. All the cell lines were tested negative for mycoplasma contamination.
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