Human SH-SY5Y neuroblastoma cells were maintained in growth media composed of Dulbecco’s Modified Eagle Medium (DMEM 1X + GlutaMAX, 4.5 g/L D-glucose, 110 mg/L sodium pyruvate; Invitrogen) containing 10% bovine growth serum (BGS; GE Life Sciences), 10 units/mL penicillin, and 10 µg/ml streptomycin. Cells were grown at 37°C with 5.5% CO2 in a humidified incubator. For reverse transfections, 5 × 104 SH-SY5Y cells in 90 µl of growth media containing no antibiotics were plated in opaque TC-treated 96-well plates containing Xfect (Clontech)/DNA transfection complexes. For each well, the transfection complexes contained 200 ng plasmid DNA of unique GLuc-ERS tail and 0.06 µl of Xfect in a final volume of 10 µl. After 28–29 h, a full media exchange into growth media containing 1.5% BGS was performed. thapsigargin (Tg; Sigma) treatments began 16–17 h after the media exchange and lasted for 8 h, a time point where we have demonstrated ERS proteins are secreted in response to thapsigargin (Trychta et al., 2018a (link)). At the end of each experiment, media was removed from the wells and cells were lyzed directly in the plate and used for luciferase assay. Cells were rinsed with 1x PBS and lyzed with 75 µl of lysis buffer [50 mM Tris (pH 7.5), 150 mM NaCl, 1% NP40, 1x protease inhibitors]. For all experiments, vehicle controls were used at a concentration equivalent to the Tg treatment.
Dmem 1x glutamax
Manufactured by Thermo Fisher Scientific
Sourced in United States, France
DMEM 1X-GlutaMAX is a cell culture medium formulation that provides essential nutrients and GlutaMAX, a stable glutamine substitute, to support the growth and maintenance of various cell types in vitro.
Automatically generated - may contain errors
Lab products found in correlation
Xfect, by Takara Bio (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
D glucose, by Thermo Fisher Scientific (1 mentions)
Thapsigargin, by Merck Group (1 mentions)
Thapsigargin tg, by Merck Group (1 mentions)
Bovine growth serum bgs, by GE Healthcare (1 mentions)
Gentamicin, by Thermo Fisher Scientific (1 mentions)
Glutamine, by Thermo Fisher Scientific (1 mentions)
Amphotericin b, by Thermo Fisher Scientific (1 mentions)
Poietics human mscs, by Lonza (1 mentions)
Non essential amino acids (neaa), by Thermo Fisher Scientific (1 mentions)
Fgm 2, by Lonza (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Fungizone antimycotic, by Thermo Fisher Scientific (1 mentions)
Mem 100x, by Thermo Fisher Scientific (1 mentions)
2 phospho l ascorbic acid trisodium salt, by Merck Group (1 mentions)
Penicillin streptomycin, by Merck Group (1 mentions)
Necrostatin 1, by Enzo Life Sciences (1 mentions)
Fetal calf serum (fcs), by Merck Group (1 mentions)
L glutamine, by Merck Group (1 mentions)
10 protocols using dmem 1x glutamax
1
Monitoring ER Stress Response in SH-SY5Y Cells
2
Transfection and Treatment of SH-SY5Y Cells
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Human SH-SY5Y neuroblastoma cells were maintained in growth media composed of Dulbecco's Modified Eagle Medium (DMEM 1X + GlutaMAX, 4.5 g/L D-glucose, 110 mg/L sodium pyruvate; Invitrogen) containing 10% bovine growth serum (BGS; GE Life Sciences), 10 units/mL penicillin, and 10 µg/mL streptomycin. Cells were grown at 37°C with 5.5% CO2 in a humidified incubator. For reverse transfections, 5 x 10 4 SH-SY5Y cells in 90 µL of growth media containing no antibiotics were plated in opaque TC-treated 96-well plates containing Xfect (Clontech)/DNA transfection complexes. For each well, the transfection complexes contained 200 ng plasmid DNA of unique GLuc-ERS tail and 0.06 µL of Xfect in a final volume of 10 µL. After 28-29h, a full media exchange into growth media containing 1.5% BGS was performed. Thapsigargin (Tg; Sigma) treatments began 16-17 h after the media exchange and lasted for 8 h. At the end of each experiment, media was removed from the wells and cells were lysed directly in the plate and used for luciferase assay. Cells were rinsed with 1x PBS and lysed with 75 µL of lysis buffer (50 mM Tris (pH 7.5), 150 mM NaCl, 1% NP40, 1x protease inhibitors). For all experiments, vehicle controls were used at a concentration equivalent to the Tg treatment.
3
Proton concentration-response of NMDA receptors
4
Culturing Caco-2 and CMT-93 Adenocarcinoma Cells
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The adenocarcinoma cell lines Caco-2 (ATCC® HTB-37) and CMT-93 (ATCC® CCL-223TM) were used for assays. Both cell lines were cultured in DMEM 1X-GlutaMAX (Gibco, Life Technologies, Grand Island, NY, USA) supplemented with 10% FBS (Gibco, Life Technologies, Grand Island, NY, USA), gentamicin (100 µg/mL) (Gibco, Life Technologies, Grand Island, NY, USA), amphotericin B (2.5 µg/mL) (Gibco, Life Technologies, Grand Island, NY, USA), and glutamine (2 nm) (Gibco, Life Technologies, Grand Island, NY, USA).
5
Maintenance of Murine Embryonic Stem Cells
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A feeder layer of inactivated murine embryonic fibroblast cells was cultured in six-well plates on gelatin (0.1%, Sigma-Aldrich G7041-100G) at a seeding density of 1.6×104 cells/cm2 in base medium consisting of DMEM (1x) +GlutaMAX™ (Gibco 10566016), 15% FBS (Gibco 10493106), 0.05 mM β-mercaptoethanol (Gibco 21985023), 1% PenStrep (100 U penicillin/ 0.1 mg/ml streptomycin, Gibco 15140122). Two h prior to seeding mES cells (from 129/Ola mice courtesy of Professor Frank Brombacher (Institute of Infectious Diseases and Molecular Medicine, Faculty of Health Sciences, University of Cape Town, Cape Town, South Africa), the medium was changed to the same base medium with the addition of CHIR99021 (Chiron, 3 µM, Sigma-Aldrich SML1046-5MG), PD0325901 (1 μM, Sigma-Aldrich PZ0162-5MG) and Leukaemia inhibitory factor (10 ng/ml, LIF, Thermo Fisher Scientific A35934). 129/Ola mES cells were seeded on the feeder layer at a density of 7×103 cells/cm2. Medium was changed daily, and cells passaged every other day.
6
Culturing and Encapsulating Human Mesenchymal Stem Cells
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Primary human mesenchymal cells (Poietics™ human MSCs, Lonza, Wlakersville. Inc.) were obtained from a commercial source and grown using the proprietary protocol provided by Lonza. Mesenchymal stem cells were cultured at cell seed densities of 2 × 106 – 4 × 106/cm2 in Dulbecco’s Modified Eagle Medium (DMEM (1X) + GlutaMAX; Gibco; Life Technologies) with 10% FBS and supplemented with penicillin and streptomycin. The growth of hMSCs for seven days preceded encapsulation. A single culture dish of hMSCs after seven days of culture contained 2 million cells at an approximately 95% confluence level. hMSCs were trypsinized and pelleted before the addition of 1 ml of a 5% alginate solution.
7
Culturing Rhinoceros Fibroblast Cell Lines
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NWRs and SWR fibroblast cell lines were obtained from the San Diego Zoo Institute for Conservation Research Frozen Zoo biobank and expanded. No IACUC approval was required for the transfer of previously established cell lines. Cells were cultured at 37°C with 5% CO2 in either Dulbecco's modified Eagle's medium (DMEM) 1X+GlutaMAX™ (Gibco; supplemented with 10% fetal bovine serum, and 1% MEM nonessential amino acids; NEAA, Gibco) or a 1:1 mix of the supplemented DMEM and Fibroblast Growth Medium (FGM™-2; Lonza). The additional proprietary growth factors and supplements (including 0.5% recombinant human fibroblast growth factor-b (rhFGF-b) and 0.05% recombinant human insulin) in the FGM-2 formulation have been shown to improve the growth of rhinoceros fibroblast cells [25 (link),26 ] and have been key to the growth and biobanking of many rhinoceros species in the Frozen Zoo. We refer to this medium as 50:50 throughout.
8
Isolation and Culture of Human Dental Pulp Stem Cells
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Stem cells were extracted from human dental pulp tissue. Patients had no systemic pathology, oral infection or disease. All patients gave their informed consent according to the Helsinki Declaration (1975), this was approved by local ethics committee (Département de la Recherche Clinique et du Développement-IDRCB n°2015-A01509-40). Pulp tissue was obtained following extraction as already described (Gronthos et al., 2000). Cells were seeded and routinely maintained in low-glucose Dulbecco’s modified Eagle’s medium [DMEM 1X-GlutaMAX™ (Gibco®)] containing 10% heat-inactivated Fetal Bovine Serum (FBS), 1% non-essential amino acids MEM 100X (Gibco®), 1% Penicillin-Streptomycin (10,000 U/mL) (Gibco®) and 0.5% Fungizone® Antimycotic (Gibco®) and 50 μg/mL 2-phospho-L-ascorbic acid trisodium salt (Sigma-Aldrich) at 37°C in a humidified 5% CO2 incubator.
9
Caco-2 and HT-29 Cell Proliferation Assays
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Caco2 and HT-29 cells were cultured in DMEM (1X) + GlutaMAX (Gibco, Cat. No. 31966-021) supplemented with 1% penicillin/streptomycin (Sigma-Aldrich, St. Louis, MO, USA, Cat. No. P4333) and 10% FCS (Sigma-Aldrich, Cat. No. F7524). For Caco2-bbe cells the medium was additionally supplemented with non-essential amino acids (- L-glutamine) (Sigma-Aldrich, Cat. No. M7145) and 100 mM HEPES (Sigma-Aldrich H0887). HCT116 cells were cultured in McCoy’s 5A medium (Sigma-Aldrich, Cat. No. M4892) supplemented with 1% penicillin/streptomycin and 10% FCS. All cell lines used in this study were obtained from the American Type Culture Collection (ATCC).
Cells were stimulated with 13.5 µM MI-2 (Selleckchem, Cat. No. S7429), 0.5 µM NSA (Merck, Darmstadt, Germany, Cat. No. 432531-71-0), 10 µM necrostatin-1 (Enzo, Farmingdale, NY, USA, Cat. No. BML-AP309), 20 µM VX-765 (Selleckchem, Cat. No. S2228), 20 µM z-VAD-FMK (MedChemExpress, Monmouth Junction, NJ, USA, Cat. No. HY-16658B), 0.2 µM liproxstatin-1 (Biomol, Hamburg, Germany, Cat. No. Cay17730) and 30 µM mepazine (MedChemExpress, Cat. No. HY-121282A). Proliferation assays were performed using the Agilent xCELLigence System or Sartorius Incucyte SX5. For Incucyte analysis cells were stained with SYTOX green nucleic acid stain (Thermo Fisher, Waltham, MA, USA, Cat. No. S7020).
Cells were stimulated with 13.5 µM MI-2 (Selleckchem, Cat. No. S7429), 0.5 µM NSA (Merck, Darmstadt, Germany, Cat. No. 432531-71-0), 10 µM necrostatin-1 (Enzo, Farmingdale, NY, USA, Cat. No. BML-AP309), 20 µM VX-765 (Selleckchem, Cat. No. S2228), 20 µM z-VAD-FMK (MedChemExpress, Monmouth Junction, NJ, USA, Cat. No. HY-16658B), 0.2 µM liproxstatin-1 (Biomol, Hamburg, Germany, Cat. No. Cay17730) and 30 µM mepazine (MedChemExpress, Cat. No. HY-121282A). Proliferation assays were performed using the Agilent xCELLigence System or Sartorius Incucyte SX5. For Incucyte analysis cells were stained with SYTOX green nucleic acid stain (Thermo Fisher, Waltham, MA, USA, Cat. No. S7020).
10
Transfection of UHRF1 Variants in HeLa Cells
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HeLa cells (ATCC, CCL-2, Amp, Cervical Adenocarcinoma; Human) and HeLa cells stably expressing either GFP-UHRF1 WT or GFP-UHRF1 C724A-H741A protein, were grown in Dulbecco's Modi ed Eagle's Medium (DMEM 1X + GlutaMAX™, Pyruvate, Gibco, Lifetech, France) which was supplemented with 10% fetal bovine serum (FBS, S1810-500, Dominique Dutscher), in addition to mixture of penicillin (100 U/ml) and streptomycin (100 U/ml) (17-602E, Lonza, USA), at 37 °C with 5% CO 2 in humidi ed environment.
Plasmids were transfected in HeLa cells with either jetPEI™ or jetPRIME (PolyPlus-transfection, France) according to the manufacturer's protocol.
Plasmids were transfected in HeLa cells with either jetPEI™ or jetPRIME (PolyPlus-transfection, France) according to the manufacturer's protocol.
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