The establishment of GRINCH cells has been described elsewhere (Haataja et al., 2013 (link)). Briefly, cDNA encoding superfolder GFP (sfGFP) was amplified and ligated into the ApaI site located at the C-peptide encoding sequence to make hPro-CpepSfGFP. Cells were cultured at 37 C in a humidified atmosphere of 95% air and 5% CO2 in RPMI 1640 culture medium containing 11 mM D-glucose (Gibco, Life Technologies) supplemented with 10% fetal bovine serum, 100 units/ml penicillin, 100 μg/mL streptomycin, 10 mM glutamine, 1 mM sodium pyruvate, 10 mm HEPES, 50 μM β-mercaptoethanol (all from Sigma) and 0.1 mg/mL G418 (CalBiochem, Germany). INS-1 (832/13) cells were cultured under the same conditions but without G418.
Rpmi 1640 culture
Manufactured by Thermo Fisher Scientific
Sourced in United States, Germany
RPMI 1640 culture is a commonly used cell culture medium formulation developed at Roswell Park Memorial Institute. It provides a balanced salt solution and nutrients required for the in vitro cultivation of a variety of cell types, including human and animal cells.
Automatically generated - may contain errors
Lab products found in correlation
β mercaptoethanol, by Merck Group (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
D glucose, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Zhejiang Tianhang Biotechnology (1 mentions)
Dulbecco s modified eagle s medium, by Thermo Fisher Scientific (1 mentions)
Non essential amino acids (neaa), by Thermo Fisher Scientific (1 mentions)
Epidermal growth factor (egf), by Merck Group (1 mentions)
Caco 2, by Thermo Fisher Scientific (1 mentions)
Caco 2, by Merck Group (1 mentions)
Ht 29, by Thermo Fisher Scientific (1 mentions)
Amphotericin b 25 μg psa, by Thermo Fisher Scientific (1 mentions)
2 mercaptoethanol, by Merck Group (1 mentions)
Fetal bovine serum (fbs), by Atlanta Biologicals (1 mentions)
Nctc 135 medium, by Merck Group (1 mentions)
Calf serum, by Thermo Fisher Scientific (1 mentions)
L glutamine, by Thermo Fisher Scientific (1 mentions)
Trypsin, by Merck Group (1 mentions)
L glutamine, by Merck Group (1 mentions)
Collagenase, by Merck Group (1 mentions)
Histopaque 1077, by Merck Group (1 mentions)
8 protocols using rpmi 1640 culture
1
Establishment and Maintenance of GRINCH Cells
2
Mouse Forestomach Carcinoma Model
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Mouse forestomach carcinoma (MFC) were purchased from the cell bank of the Chinese Academy of Sciences. RPMI-1640 culture was purchased from the Gibco of the United States. Fetal bovine serum was purchased from Zhejiang Tianhang Biotechnology Co. Ltd. Trypsin, penicillin and streptomycin were purchased from Shanghai Genom Biotechnology Co. Ltd. The mouse Nesfatin-1 Elisa Kit (CEA242Mu) was purchased from Wuhan Youo Technology Development Co. Ltd. Rabbit anti-Nesfatin-1 polyclonal antibody was purchased from the Sigma Company. Horseradish peroxidase (HPR) tagged goat anti-rabbit secondary antibody was purchased from the Biosharp Company.
3
Culturing LOVO and iBMDM Cells
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Mouse iBMDM and human colon carcinoma cells (LOVO) were obtained via commercial approaches and used for in vitro experiments. LOVO cells were cultured in Dulbecco's modified Eagle's medium (Gibco, USA) supplemented with 10% fetal bovine serum (FBS, Gibco, USA). IBMDM cells were cultured in RPMI 1640 culture (Gibco, USA) containing 10% FBS. LOVO and iBMDM were cultured in an incubator with a constant temperature of 37 °C with 5% CO2.
4
Colorectal Adenocarcinoma Cell Culture
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The human epithelial colorectal adenocarcinoma cell lines HT-29 and Caco-2 were acquired from the Kunming Cell Bank of Type Culture Collection, Chinese Academy of Sciences (Kunming, Yunan, China). HT-29 and Caco-2 cells were cultured in RPMI 1604 medium (Gibco, Grand Island, NY, USA) and Dulbecco’s modified Eagle’s medium (DMEM) (Hyclone, Logan, UT, USA), respectively. Both media contained 10% fetal bovine serum (FBS), 1 mM pyruvate and 1 × NEAA (cat #11140050, Gibco).
Differentiated HT-29 cells were cultured as described previously, with minor modifications20 (link). Briefly, glucose and pyruvate-free RPMI 1640 culture medium (cat #11879-020, Gibco) with 10% FBS was repeatedly supplemented with 20 mM glucose or 5 mM galactose for HT-29 culture. The numbers of dead cells did not significantly increase after transfer from normal RPMI 1640 medium to the indicated glucose or galactose medium. Cell-based assays were carried out after at least 5 days of cultivation. For EGF treatment, Caco-2 cells were pre-starved in serum-free medium for 10 hr and then stimulated with EGF (200 ng/ml; Millipore, Bedford, MA, USA).
Differentiated HT-29 cells were cultured as described previously, with minor modifications20 (link). Briefly, glucose and pyruvate-free RPMI 1640 culture medium (cat #11879-020, Gibco) with 10% FBS was repeatedly supplemented with 20 mM glucose or 5 mM galactose for HT-29 culture. The numbers of dead cells did not significantly increase after transfer from normal RPMI 1640 medium to the indicated glucose or galactose medium. Cell-based assays were carried out after at least 5 days of cultivation. For EGF treatment, Caco-2 cells were pre-starved in serum-free medium for 10 hr and then stimulated with EGF (200 ng/ml; Millipore, Bedford, MA, USA).
5
Cellular Uptake of Doxorubicin-Loaded Nanostructures
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The in vitro cellular uptake
of the developed nanostructures DOX@NMOF, DOX@NMOF-PEI, and DOX@NMOF-PEI-GA
in addition to the free DOX was investigated by the immunofluorescence
microscopy using HepG2 cells. HepG2 cells were cultured using RPMI-1640
culture (Gibco, ThermoScientific, Germany), treated with 10% fetal
bovine serum (FBS) (Gibco, ThermoScientific, Germany) and 1% of penicillin
G sodium salt (10,000 UI), streptomycin (10 mg), and amphotericin
B (25 μg) (PSA) (Gibco, ThermoScientific, Germany). The cells
were incubated for 24 h, and then the tested formulations were added
using their IC50 values obtained at 24 h. The cellular
uptake was examined by a LABOMED fluorescence microscope LX400, cat
no: 9126000. Briefly, the cells were fixed with cold methanol and
then stained with DAPI. The images were generated by OptikaI Sveiw
software by applying 2 filters: the first one is the drug that emits
a red color, and the excitation/emission were 470/595, respectively,
and the second filter was DAPI, which emits a blue color, and the
excitation/emission were 340/452, respectively. Violet color was produced
upon the intersection of the red and blue colors.
of the developed nanostructures DOX@NMOF, DOX@NMOF-PEI, and DOX@NMOF-PEI-GA
in addition to the free DOX was investigated by the immunofluorescence
microscopy using HepG2 cells. HepG2 cells were cultured using RPMI-1640
culture (Gibco, ThermoScientific, Germany), treated with 10% fetal
bovine serum (FBS) (Gibco, ThermoScientific, Germany) and 1% of penicillin
G sodium salt (10,000 UI), streptomycin (10 mg), and amphotericin
B (25 μg) (PSA) (Gibco, ThermoScientific, Germany). The cells
were incubated for 24 h, and then the tested formulations were added
using their IC50 values obtained at 24 h. The cellular
uptake was examined by a LABOMED fluorescence microscope LX400, cat
no: 9126000. Briefly, the cells were fixed with cold methanol and
then stained with DAPI. The images were generated by OptikaI Sveiw
software by applying 2 filters: the first one is the drug that emits
a red color, and the excitation/emission were 470/595, respectively,
and the second filter was DAPI, which emits a blue color, and the
excitation/emission were 340/452, respectively. Violet color was produced
upon the intersection of the red and blue colors.
6
Rcho-1 TS Cell Differentiation Model
7
Chromosome Analysis of Proband's Family
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G-banded chromosome analysis were performed using cultured peripheral blood lymphocytes from the proband and her parents. Two milliliters of peripheral blood samples were collected with heparin added and subjected to RPMI1640 culture medium with 20% calf serum (Invitrogen Gibco, USA) at 37 °C in 5% CO2. Preparation of metaphase and conventional cytogenetics followed standard cytogenetic protocols. A minimum of 20 metaphases with good chromosome separation were analyzed. Karyotypes were described according to the International System for Human Cytogenetic Nomenclature (International Standing Committee on Human Cytogenetic Nomenclature, 2009).
8
Isolation and Culture of Pancreatic Islets
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Sprague-Dawley rats (8 to 10 weeks old, body weight 250 to 300 g, provided by the Guangxi Medical University Laboratory Animal Center) were sacrificed, and collagenase (Sigma) was injected into the ductal system for tissue digestion. 16 The tissue was dispersed under shaking after collagenase digestion. The islets were isolated through density-gradient centrifugation using Histopaque-1077 (Sigma). The islets were dispersed into single cells via trypsin (Sigma) digestion. The cells were plated and grown in culture dishes containing 40-mm-diameter coverslips overnight in Roswell Park Memorial Institute 1640 (RPMI 1640) culture medium (Invitrogen) supplemented with 10% (vol∕vol) fetal calf serum, 100 IU∕ml penicillin, 0.1 mg∕ ml streptomycin, and 5.6 mmol∕l glucose, under environmental conditions of 37°C and 5% CO 2 .
INS-1 insulinoma cells (from the Conservation Genetics Chinese Academy of Sciences Kunming Cell Bank, China, Kunming) were cultured in RPMI 1640 with 2.05 mmol∕l L-glutamine (Invitrogen) supplemented with 10% fetal calf serum, 100 IU∕ml penicillin, 0.1 mg∕ml streptomycin, 2 mmol∕l L-glutamine, 1 mmol∕l sodium pyruvate, and 50 μmol∕l b-mercaptoethanol (Sigma). Sterile conditions were maintained for all operations involving islet isolation and cell culture. All cells were attachment cultured in the FCS2 system.
INS-1 insulinoma cells (from the Conservation Genetics Chinese Academy of Sciences Kunming Cell Bank, China, Kunming) were cultured in RPMI 1640 with 2.05 mmol∕l L-glutamine (Invitrogen) supplemented with 10% fetal calf serum, 100 IU∕ml penicillin, 0.1 mg∕ml streptomycin, 2 mmol∕l L-glutamine, 1 mmol∕l sodium pyruvate, and 50 μmol∕l b-mercaptoethanol (Sigma). Sterile conditions were maintained for all operations involving islet isolation and cell culture. All cells were attachment cultured in the FCS2 system.
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