Mouse cortices were isolated from postnatal day 1 mouse pups (Chen et al., 2007 (link)) in accordance with the NIH Guide for the Care and Use of Laboratory Animals. Cortical tissues were diced into ∼1 mm3 pieces in a 60 mm dish with a sterilized razor blade, and the minced tissues were resuspended with freshly prepared D/F20S medium: Dulbecco’s Modified Eagle Medium: Nutrient Mixture F-12 (DMEM/F-12; Gibco, Grand Island, NY, USA) supplemented with 20% v/v FBS (HyClone, Logan, UT, USA) and 1% v/v Penicillin/streptomycin (P/S; Gibco; 5 ml D/F20S medium added for each brain). Tissues were then pipetted up and down until homogenized, transferred into flasks (material from one brain to one T25 flask; Corning) and incubated with 5% CO2 at 37°C. Seventy-two hours after plating medium was replaced, most cortical pieces attached onto flasks and some flat cells started to migrate out of tissues. Afterward, medium was replaced every other day.
Nutrient mixture f 12 dmem f 12
Manufactured by Thermo Fisher Scientific
Sourced in United States
Nutrient Mixture F-12 (DMEM/F-12) is a cell culture medium designed for the growth and maintenance of a variety of cell types. It is a balanced salt solution that provides essential nutrients, vitamins, and amino acids required for cell proliferation and survival.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (12 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (9 mentions)
Epidermal growth factor (egf), by Thermo Fisher Scientific (6 mentions)
Hydrocortisone, by Merck Group (5 mentions)
Streptomycin, by Thermo Fisher Scientific (4 mentions)
L glutamine, by Thermo Fisher Scientific (4 mentions)
Penicillin, by Thermo Fisher Scientific (3 mentions)
Basic fibroblast growth factor (bfgf), by Thermo Fisher Scientific (3 mentions)
Neurobasal a medium, by Thermo Fisher Scientific (3 mentions)
Non essential amino acid (neaa), by Thermo Fisher Scientific (2 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (2 mentions)
Insulin, by Merck Group (2 mentions)
N2 supplement, by Thermo Fisher Scientific (2 mentions)
Cholera toxin, by Merck Group (2 mentions)
Penicillin streptomycin solution, by Thermo Fisher Scientific (2 mentions)
T25 flask, by Corning (1 mentions)
Penicillin streptomycin p s, by Thermo Fisher Scientific (1 mentions)
Fungizone, by Thermo Fisher Scientific (1 mentions)
Fetal clone 1 hyclone, by Thermo Fisher Scientific (1 mentions)
Collagenase type 2, by Worthington (1 mentions)
38 protocols using nutrient mixture f 12 dmem f 12
1
Primary Mouse Cortical Cell Culture
2
Isolation of Primary Human Osteoblasts
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Primary human osteoblasts were isolated from redundant trabecular bone fragments obtained from healthy donors undergoing pre-implant bony reconstruction of the mandible or maxilla with autologous bone from the anterior iliac crest. The donor group consisted of 11 males and 12 females with a mean age of 49.3±18.6 years. The protocol was approved by the Medical Ethical Review Board of the VU University Medical Center, Amsterdam, the Netherlands, and all donors gave their written informed consent.
A modification of the methods of Beresford and Marie [18] , [19] (link) was used. Shortly, the trabecular bone fragments were minced into small pieces and washed extensively with phosphate buffered saline (PBS). The bone pieces were treated with 2 mg/ml collagenase type II (300 U/mg; Worthington Biochemical Corporation, Lakewood, NJ, USA) for two hours in a shaking waterbath at 37°C. The pieces were placed in culture flasks with Dulbecco’s Modified Eagle Medium: Nutrient Mixture F-12 (DMEM/F12; GIBCO, Life technologies) supplemented with 10% Fetal Clone I (HyClone, Thermo Fisher Scientific), 100 U/ml penicillin and 100 µg/ml streptomycin (GIBCO, Life technologies), 1.25 µg/ml fungizone (GIBCO, Life technologies) and incubated at 37°C and 5% CO2. Medium was changed twice a week until cells reached confluence.
A modification of the methods of Beresford and Marie [18] , [19] (link) was used. Shortly, the trabecular bone fragments were minced into small pieces and washed extensively with phosphate buffered saline (PBS). The bone pieces were treated with 2 mg/ml collagenase type II (300 U/mg; Worthington Biochemical Corporation, Lakewood, NJ, USA) for two hours in a shaking waterbath at 37°C. The pieces were placed in culture flasks with Dulbecco’s Modified Eagle Medium: Nutrient Mixture F-12 (DMEM/F12; GIBCO, Life technologies) supplemented with 10% Fetal Clone I (HyClone, Thermo Fisher Scientific), 100 U/ml penicillin and 100 µg/ml streptomycin (GIBCO, Life technologies), 1.25 µg/ml fungizone (GIBCO, Life technologies) and incubated at 37°C and 5% CO2. Medium was changed twice a week until cells reached confluence.
3
Cell Culture Media Preparation
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All materials used were of analytical grade. Dulbecco’s Modified Eagle Medium: Nutrient Mixture F-12 (DMEM/F-12), fetal calf serum (FCS) and gentamycin were obtained from Gibco (USA).
4
Generating Imatinib-Resistant Leukemia Cell Line
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The human leukemia K562 cell line (CML) was kindly provided by Dr. Kai-Wen Hsu, Graduate Institute of New Drug Development and Biomedical Sciences, China Medical University, Taichung, Taiwan. Imatinib-resistant K562 (IR-K562) cells were originally derived from K562 cells by treatment with 0.05 μM imatinib for 2 months and treatment with 0.1, 0.5, 1 and 5 μM imatinib for one month. While training IR-K562 cells, we changed the culture medium every week. The cells were maintained in Dulbecco’s modified Eagle’s medium: Nutrient Mixture F-12 (DMEM/F-12) (Gibco, Carlsbad, CA, USA). The cells were incubated with 10% (v/v) fetal bovine serum (FBS, Biological Industries, Israel). Supplements of 100 units/mL penicillin and 100 mg/mL streptomycin were used and cultured in a 37 °C incubator with 5.0% CO2.
5
Glioma Initiating Cells Characterization
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Both GICs belong to non-mutated and non-G-CIMP (G-CIMP-) subtypes. Figure 1 displays the optical microscopy images in the medium with and without laminin coated surface. GICs grow as neurospheres in non-laminin coated plates and in adherence in laminin coated surface. Cells placed on 7,5 mg/mL laminin-coated plates (Sigma, St. Louis, MO, USA) were maintained in a complete Neuronal Stem Cell (NSC) medium at 37 °C in a humidified 5% CO2 and 5% O2 atmosphere (hypoxia conditions) to simulate brain microenvironment (Heracell 150i incubator). NSC medium was constituted by Dulbecco’s Modified Eagle Medium and Nutrient Mixture F-12,DMEM/F12, (Gibco, Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with N2 (GIBCO), 4,5% glucose (Sigma, Merck KGaA, Darmstadt, Germany), 1M Hepes (Sigma), 2% BSA (Sigma) basic fibroblast growth factor 20 ng/mL (Gibco), and epidermal growth factor 20 ng/mL (Gibco).
GICs were treated with 200 µM or 2mM of Na[o-COSAN] to study the uptake of the anionic small molecule [o-COSAN]− and its effects on cell function.
Glioma initiating cells (GICs), proneural GIC7 (a kind gift from Dr. Marta María Alonso, Department of Pediatrics, Clínica Universidad de Navarra, University of Navarra, Pamplona, Spain) [74 (link)], and mesenchymal PG88 cells were obtained from human GBM specimens as described previously [46 (link),75 (link)].
GICs were treated with 200 µM or 2mM of Na[o-COSAN] to study the uptake of the anionic small molecule [o-COSAN]− and its effects on cell function.
Glioma initiating cells (GICs), proneural GIC7 (a kind gift from Dr. Marta María Alonso, Department of Pediatrics, Clínica Universidad de Navarra, University of Navarra, Pamplona, Spain) [74 (link)], and mesenchymal PG88 cells were obtained from human GBM specimens as described previously [46 (link),75 (link)].
6
Culturing Colorectal Cancer Cell Lines
7
Murine Pancreatic Acini Isolation
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Six-week-old healthy CD-1 mice were euthanized, and the pancreatic tissues were collected. Minced pancreatic tissues were dispersed in DMEM with Nutrient Mixture F-12 (DMEM/F12, Gibco) supplemented with 1.0 U/ml collagenase D from Clostridium histolyticum and 0.25 mg/ml trypsin inhibitor from soybean (both from Roche Diagnostics). The pancreatic tissues were vigorously shaken at 200 rpm for 2 h at 37 °C. Digested tissue was filtered by using a cell strainer (100 μm pore size, Corning). Pancreatic acini retained by the cell strainer were transferred to a 24-well tissue culture plate and cultured overnight in DMEM/F12 medium supplemented with 5% FBS and 1% penicillin-streptomycin. Non-adherent pancreatic acini were then transferred into a new 48-well tissue culture plate and used immediately, while adherent contaminant cells were discarded.
8
Neural Stem Cell Proliferation and Differentiation
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NSCs were harvested from rats following a previously described protocol (Lu et al., 2012a, b; Li et al., 2014). The original NSC culture medium was proliferation medium plus 50 IU/mL penicillin and 50 μg/mL streptomycin. Pure proliferation of NSCs was induced by 10 ng/mL basic fibroblast growth factor (Promega, Madison, WI, USA) and 10 ng/mL epidermal growth factor (Promega) in basic culture medium, which contained Dulbecco's Modified Eagle Media: Nutrient Mixture F12 (DMEM/F12, 1:1, v/v, Gibco, Grand Island, NY, USA), 2% B27 (Gibco), 1% N2 (Gibco) supplement, 0.5 mM L-glutamine, and 0.5 mM non-essential amino acid (Gibco). Pure differentiation culture medium mainly contained 1% fetal bovine serum (Gibco) and 1% serum replacement (Gibco) in basic culture medium without basic fibroblast growth factor and epidermal growth factor (Vescovi and Snyder, 1999; Wen et al., 2002, 2007; Yu et al., 2007; Lu et al., 2012a, b; Li et al., 2014; SanMartin et al., 2014). In each treatment group, different concentrations of GM1 and/or propofol combined with remifentanil were added into pure proliferation or differentiation culture medium at the beginning, and co-cultured during the entire proliferation or differentiation process.
9
Culturing A549 and Caco-2 Cell Lines
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The non-small-cell human lung carcinoma A549 cells were obtained from American Type Culture Collection (Rockville, MD, USA). Caco-2 human corectal adenocarcinoma cells were obtained from Dr. Iliyan IIiev’s laboratory (Institute for Research in IBD, Weill Cornell Medicine of Cornell University). Cells were maintained in Dulbecco’s Modified Eagle Medium/Nutrient Mixture F-12 (DMEM/F-12) (Gibco, Waltham, MA, USA) supplemented with 10% fetal bovine serum (10% FBS) (Gibco, Waltham, MA, USA) and 100 U/mL penicillin and 100 μg/mL streptomycin (P/S). Cells were cultured at 37 °C humidified atmosphere containing 5% of CO2. Cells were fed every 2–3 days and subculture upon reaching 70–80% confluence.
10
Isolation and Culture of Human Luteinized Granulosa Cells
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Ovarian stimulation and oocyte retrieval were performed under a gonadotropin-releasing hormone (GnRH) antagonist or agonist protocol. After adequate follicle development, as detected by both ovarian ultrasound and serum estradiol assay, human chorionic gonadotropin (Lvzhu, Zhuhai, China) was administered to trigger ovulation. Ultrasound-guided oocyte retrieval was performed 36 h later.
Human luteinized granulosa cells (hLGCs) were retrieved from the follicular fluid as previously described [43 (link)]. The follicular fluid was pooled and centrifuged at 2500 rpm for 15 min, then the pellets were re-suspended in phosphate-buffered saline (PBS) and dispersed in 0.1% hyaluronidase (Sigma Chemical Co., St. Louis, MO, USA) at 37 °C for 15 min. Granulosa cells then were purified by Ficoll-Paque (GE Healthcare Bio-Science, Uppsala, Sweden). The isolated granulosa cells were stored at −80 °C or used after 3 days in culture. SVOG and KGN cells were obtained from Shandong University. All the hLGCs and SVOG and KGN cells were cultured in Dulbecco's Modified Eagle Medium: Nutrient Mixture F-12 (DMEM/F12) (Gibco, Grand Island, NY) containing 10% charcoal-stripped fetal bovine serum (Biological Industries, US origin) and 1% penicillin-streptomycin-neomycin (PSN, Gibco) at 37 °C in a humidified atmosphere with 5% CO2. KGN and SVOG cells were passaged every 3 days.
Human luteinized granulosa cells (hLGCs) were retrieved from the follicular fluid as previously described [43 (link)]. The follicular fluid was pooled and centrifuged at 2500 rpm for 15 min, then the pellets were re-suspended in phosphate-buffered saline (PBS) and dispersed in 0.1% hyaluronidase (Sigma Chemical Co., St. Louis, MO, USA) at 37 °C for 15 min. Granulosa cells then were purified by Ficoll-Paque (GE Healthcare Bio-Science, Uppsala, Sweden). The isolated granulosa cells were stored at −80 °C or used after 3 days in culture. SVOG and KGN cells were obtained from Shandong University. All the hLGCs and SVOG and KGN cells were cultured in Dulbecco's Modified Eagle Medium: Nutrient Mixture F-12 (DMEM/F12) (Gibco, Grand Island, NY) containing 10% charcoal-stripped fetal bovine serum (Biological Industries, US origin) and 1% penicillin-streptomycin-neomycin (PSN, Gibco) at 37 °C in a humidified atmosphere with 5% CO2. KGN and SVOG cells were passaged every 3 days.
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