HEK293T cells (ATTC; CRL-3216) and wt or ko diaphragm-derived cells (DDCs) derived from C57BL/6 and CIM mice21 (link) were maintained by serial passage in Dulbecco's modified Eagle's medium, high glucose (Invitrogen Life Technologies) supplemented with 2 mM l -glutamine, 1 mM sodium pyruvate, 1× Minimum Essential Medium non-essential amino acids, 100 U ml−1 penicillin, 100 mg ml−1 streptomycin (PAA) and 10% fetal calf serum (FCS, Biochrom). Human foreskin fibroblasts (HS27, ATCC; CRL-1634) were maintained in Iscove's modified Dulbecco's medium, high glucose (Invitrogen Life Technologies) supplemented with 100 U ml−1 penicillin, 100 mg ml−1 streptomycin and 5% FCS. All cells were mycoplasma-free and regularly tested by PCR61 (link).
High glucose
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High glucose is a laboratory reagent used for the quantitative determination of glucose concentration in biological samples. It provides a precise and reliable method for measuring glucose levels.
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Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (56 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (40 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (27 mentions)
Penicillin, by Thermo Fisher Scientific (17 mentions)
Streptomycin, by Thermo Fisher Scientific (16 mentions)
L glutamine, by Thermo Fisher Scientific (12 mentions)
Glutamine, by Thermo Fisher Scientific (7 mentions)
Pyruvate, by Thermo Fisher Scientific (7 mentions)
Non essential amino acids (neaa), by Thermo Fisher Scientific (6 mentions)
Sodium pyruvate, by Thermo Fisher Scientific (6 mentions)
Fugene hd, by Promega (6 mentions)
Glutamax, by Thermo Fisher Scientific (5 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (5 mentions)
Dulbecco s modified eagle s medium, by Thermo Fisher Scientific (5 mentions)
Glutamax supplement, by Thermo Fisher Scientific (5 mentions)
Dmem medium, by Thermo Fisher Scientific (5 mentions)
Penicillin, by Merck Group (4 mentions)
Fetal bovine serum (fbs), by Merck Group (4 mentions)
Penicillin streptomycin, by Merck Group (4 mentions)
Fetal calf serum (fcs), by Thermo Fisher Scientific (3 mentions)
170 protocols using high glucose
1
Maintenance of Cell Lines for Research
2
Cell Culture Protocols for Fibroblasts and Macrophages
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L929 mouse fibroblasts and wt or Irga6 ko MEFs derived from C57BL/6 mice were maintained by serial passage in Dulbecco's modified Eagle medium (DMEM), high glucose (Invitrogen Life Technologies) supplemented with 2 mM of l ‐glutamine, 1 mM of sodium pyruvate, 1× minimal essential medium non‐essential amino acids, 100 U ml−1 of penicillin, 100 mg ml−1 of streptomycin (PAA) and 10% fetal calf serum (FCS, Biochrom). C57BL/6 BMMs were cultured in DMEM, high glucose containing 10% L929 P2 cell‐conditioned medium and supplements indicated earlier. IRG protein expression was induced with 200 U ml−1 of IFNγ (Cell Concepts) for 24 h before infection. Human foreskin fibroblasts (HS27, ATCC CRL‐1634) were maintained in Iscove's modified Dulbecco's medium, high glucose (Invitrogen Life Technologies) supplemented with 100 U ml−1 of penicillin, 100 mg ml−1 of streptomycin and 5% FCS.
3
Culturing FaDu and Rat Macrophages for Nanoshell Study
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FaDu cells (human head and neck squamous cell carcinoma: HNSCC, ATCC, and HTB-43) were grown in Dulbecco's Modified Eagle Medium (DMEM, Gibco, Carlsbad, CA) with high glucose (Invitrogen Corp., Carlsbad, CA) supplemented with 10% fetal bovine serum (FBS, Gibco, Carlsbad, CA), 2 mM L-glutamine, and 100 mg/ml gentamycin. Rat alveolar macrophages (NR8383; ATCC, CRL-2192) were maintained in DMEM with high glucose supplemented with 10% FBS, 25 mM HEPES buffer (Gibco, Carlsbad, CA) (pH 7.4), 100 U/ml penicillin, and 100 mg/ml streptomycin. All cultures were maintained at 37°C and 7.5% CO2. The gold nanoshells (ANS, Nanospectra Biosciences, Inc., Houston, TX) used in this study consist of a 120 nm diameter silica core surrounded by a 12 to 15 nm thick gold shell. The nanoshells are PEGylated to inhibit aggregation and have peak absorption between 800 and 810 nm (Figure 1(b) ).
4
Cultivation and Maintenance of RAW264.7 and Caco-2 Cells
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RAW264.7 and human epithelial colorectal adenocarcinoma (Caco-2) cells were provided by the Cell Resource Center of the Shanghai Institute for Biological Science, Chinese Academy of Sciences, China. RAW264.7 cells were cultured in Dulbecco’s modified Eagle’s medium (high glucose, Gibco®, Thermo Fisher Scientific, Waltham, MA, USA), supplemented with 10% fetal bovine serum (Premium, Pan Biotech, Adenbach, Germany and 1% pen–strep antibiotics (GibcoTM, Thermo Fisher Scientific, Waltham, MA, USA) and then incubated in a humidified atmosphere of 5% CO2 at 37 ℃. Caco-2 cells were cultured in Dulbecco’s modified Eagle’s medium (high glucose, Gibco®, Thermo Fisher Scientific, Waltham, MA, USA), supplemented with 20% fetal bovine serum (Premium, Pan Biotech, Adenbach, Germany) and 1% pen–strep antibiotics (GibcoTM, Thermo Fisher Scientific, Waltham, MA, USA) and then incubated in a humidified atmosphere of 5% CO2 at 37 ℃.
5
Generating Stable Cell Lines with Lsm10 and Lsm11
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HEK293T and NIH3T3 cells were cultured under 5%CO2 at 37°C in Dulbecco’s modified Eagle’s medium with high glucose (Invitrogen) containing 10% of fetal bovine serum (HyClone), 2 mM glutamine (Gibco), and 10 μg/mL Gentamicin (Gibco). The NIH3T3-SmnRNAi cell line used in this study was described previously (Lotti et al., 2012 (link); Ruggiu et al., 2012 (link)). To generate stable NIH3T3 cell lines with overexpression of Lsm10 and/or Lsm11, NIH3T3-SmnRNAi cells were transduced with the corresponding lentiviral vectors and selected with neomycin/G418 (0.5 mg/mL; for NIH3T3-Lsm10/SmnRNAi), Hygromycin B (250 μg/mL; for NIH3T3-Lsm11/SmnRNAi) or both antibiotics (for NIH3T3-Lsm10/Lsm11/SmnRNAi). Depletion of endogenous Smn was induced by treatment with doxycycline (100 ng/mL) for 5 days prior to analysis. All cell lines tested negative for mycoplasma contamination.
6
Cultivation of Hybrid EPEC/ETEC Isolates
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The LT+ (102712, 102771, and 102651) and EatA+ (401140) EPEC/ETEC hybrid isolates examined in this study were isolated through the Global Enteric Multisite Study (GEMS)25 (link). The EPEC/ETEC isolates were grown in Lysogeny Broth (LB)54 (link) (Difco), or in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with high glucose (4.5 g/L) or low glucose (1 g/L)(Invitrogen). Bile salts were supplemented at 3% (wt/vol) in described media.
7
Culturing Swine Testicle and HEK-293 Cells
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Swine testicle (ST) cells and human embryonic kidney (HEK-293) cells were cultured in Dulbecco’s modified Eagle medium (DMEM) with high glucose (Invitrogen) supplemented with 10 % fetal bovine serum (FBS, Invitrogen) and antibiotic–antimycotic solutions(×100; Invitrogen). The cells were maintained at 37 °C in 5 % CO2. Eukaryotic expression vector, pVAX1, was stored at −20 °C in our laboratory. The TGEV strain SC-H was isolated from Sichuan, P.R. China in 2005. The attenuated S. typhimurium aroA-strain SL7207 was kindly provided by Professor Kai Schulze of Helmholtz Centre for Infection Research, Germany.
8
Chondrogenic Differentiation of MSCs
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MSCs were trypsinized and seeded in V-bottomed 96-well polypropylene plates at 200,000 cells per well. Pellets were formed by centrifugation at 250 g for 5 min and chondrogenically differentiated in DMEM with high glucose (Invitrogen), 1% ITS (Sigma Aldrich), 50 μg/ml ascorbate-2-phosphate (Sigma Aldrich), 40 μg/ml L-proline (Sigma Aldrich), 100 nM dexamethasone (Sigma Aldrich), 1 mM sodium pyruvat (Invitrogen) and 10 ng/ml TGFβ1 (R&D Systems).
After a pre-differentiation period of 14 days, medium conditions were changed to hypertrophy enhancing medium (hyp) consisting of DMEM high glucose, 1% ITS, 50 g/ml ascorbate-2-phosphate, 40 g/ml L-proline, 1 nM triiodothyronine (T3) (Sigma Aldrich) and the control was kept in chondrogenic medium (chon) for the whole culture period. The medium was changed three times per week.
Aggregates were harvested at d1, d3, d7, d14, d17, d21 and d28 for gene expression analysis. Aggregates for histological analysis were harvested on d14 and d28.
After a pre-differentiation period of 14 days, medium conditions were changed to hypertrophy enhancing medium (hyp) consisting of DMEM high glucose, 1% ITS, 50 g/ml ascorbate-2-phosphate, 40 g/ml L-proline, 1 nM triiodothyronine (T3) (Sigma Aldrich) and the control was kept in chondrogenic medium (chon) for the whole culture period. The medium was changed three times per week.
Aggregates were harvested at d1, d3, d7, d14, d17, d21 and d28 for gene expression analysis. Aggregates for histological analysis were harvested on d14 and d28.
9
Cell Culture Protocol for Glioblastoma and Breast Cancer
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U87-EGFRvIII cells were cultured in DMEM (Dulbecco’s modified Eagle’s medium) with high glucose (Invitrogen), 10% fetal bovine serum (FBS), 1% streptomycin–penicillin, 1% Glutamax (Invitrogen), and hygromycin B (30 μg/mL), while MDA-MB-231 and MCF-7 cells were cultured in DMEM/F-12 with 10% FBS, 1% streptomycin–penicillin, and 1% Glutamax.
10
Adipogenesis in 3T3-L1 Murine Preadipocytes
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3T3-L1 murine preadipocytes were purchased from ATCC (ATCC, Manassas, VA). After thawing, 3T3-L1 cells were cultured at 37°C in a 5% CO2 incubator in α-minimal essential medium (α-MEM, Invitrogen, Carlsbad CA) supplemented with 10% heat inactivated fetal bovine serum (FBS, Invitrogen, Carlsbad, CA) and 1% antibiotic/antimycotic solution (Invitrogen, Carlsbad, CA). The medium was changed after 48 h and every 3-4 days thereafter as described previously [10 (link)]. For adipogenesis studies the medium was replaced with adipogenic medium (Dulbecco's modified Eagle Medium (DMEM)) with high glucose (Invitrogen), supplemented with 10% (v/v) FBS, 10 μg/ml insulin (Sigma-Aldrich, St. Louis, MO), 0.5 mM dexamethasone (Sigma-Aldrich), and 0.1 mM indomethacin (Sigma-Aldrich) and the cells were cultured for an additional 8 days. Cells were cultured in the absence and presence of EET-A at a dose of 10 μM. At the experimental endpoints, cells were collected by trypsinization, washed once with PBS, and then lysed for protein measurements and for RNA extraction.
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