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Distribution and Toxicity of Retroviral Vectors after Intracavitary Delivery in Mouse and Man
Published in Eric Wickstrom, Clinical Trials of Genetic Therapy with Antisense DNA and DNA Vectors, 2020
Patrice S. Obermiller, Carlos L. Arteaga, Jeffrey T. Holt, Anne M. Pilaro
The retroviral vector producer cell line PA317 was grown in DMEM high glucose (Life Technologies) with L-glutamine, 10% fetal bovine serum (Hyclone) and antibiotic/antimycotic (Sigma) added. When in the artificial capillary system for mass production, the antibiotic/antimycotic was changed to a penicillin/streptomycin solution (Sigma), and the temperature was reduced to 34°C. Before injection into mice, cells were scraped (not trypsinized) off the plates, pelleted and resuspended in warm medium to an appropriate density.
Lysosomal Vitamin B12 Trafficking
Published in Bruno Gasnier, Michael X. Zhu, Ion and Molecule Transport in Lysosomes, 2020
Sean Froese, Matthias R. Baumgartner
Human adherent cells (e.g. fibroblasts, HEK293) with or without treatment (e.g. transfection, small molecule incubation) are cultured in T25cm2 culture flasks (TPP) in routine culture medium consisting of Dulbecco’s Modified Eagle’s Medium (DMEM/F12 + GlutaMax™, Gibco) supplemented with 10% (v/v) foetal calf serum (FCS, Gibco) and 1% antibiotic-antimycotic mixture (penicillin 10,000 IU/mL, streptomycin 10,000 µg/mL, amphotericin B 25 µg/mL, Gibco). Concurrently, in the dark or under a red safelight (Dr. Fischer), cyano-[57Co]-Cbl (stock 10.5 µCi and 25 pg/mL, ICN Pharmaceuticals) is added to normal human serum (1 µL [57Co]-B12 Tracer Stock/mL serum), covered with aluminium foil and pre-incubated for 30 min at 37°C to bind the labelled cobalamin to human transcobalamin. This serum is then added to DMEM/F12 + GlutaMax supplemented with 1% antibiotic-antimycotic (final concentration, 1:10 serum to medium) and filter-sterilized. Upon reaching 80–100% confluence, cells are first washed with 5 mL DMEM/F12 + GlutaMax to remove the FCS, then each flask is incubated with 2 mL of the filter-sterilized radio-labelled medium. Incubation takes place in a cell incubator at 37°C supplemented with 5% CO2 for three days while covered in aluminium foil to prevent light exposure.
Hesperidin-loaded nanoemulsions improve cytotoxicity, induce apoptosis, and downregulate miR-21 and miR-155 expression in MCF-7
Published in Journal of Microencapsulation, 2021
Judie Magura, Daniel Hassan, Roshila Moodley, Irene Mackraj
Cell culture, dimethyl sulfoxide (DMSO), 3–(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT), polyethylene glycol 2000 (PEG-2000), Tween® 80, and hesperidin were purchased from Sigma-Aldrich, Germany. Foetal bovine serum (FBS), Dulbecco's phosphate-buffered saline (DPBS), Dulbecco's modified Eagle's medium (DMEM), and trypsin were obtained from Biowest, USA. Antibiotic-Antimycotic was acquired from Gibco by Life Technologies. Fresh sheep blood was purchased from Polychem Handelsges (South Africa). The kits used for the apoptosis and cell cycle analysis were obtained from BD Pharmingen™, California San Jose, USA. Purelink miRNA isolation kit (Thermo Fischer Scientific, USA), SensiFast™ cDNA synthesis kit (Bioline, USA), SYBR green (Roche Diagnostics, IN, USA), and specific primers were synthesised from Inqaba Biotec™ laboratories, South Africa. All other reagents and solvents were of analytical grade.
Tumor regression and immunity in combination therapy with anti-CEA chimeric antigen receptor T cells and anti-CEA-IL2 immunocytokine
Published in OncoImmunology, 2021
Seung E. Cha, Maciej Kujawski, Paul J. Yazaki, Christine Brown, John E. Shively
Mock or anti-CEA CAR T cells were incubated with either MC38/GFP, MC38/CEA/GFP, E0771/GFP, or E0771/CEA/GFP cells at 0.1:1, 0.5:1, 1:1, 2:1, 5:1, or 10:1 ratio (Effectors:Targets, E:T) in RPMI 1640 medium, no phenol red (Gibco, 11835030) with 10% FBS, 2 mM L-glutamine, antibiotic-antimycotic solution, and β-mercaptoethanol on 96 wells (Eppendorf, 951040145) overnight at 37°C. For controls, only target cells were plated. For immunocytokine (ICK) studies, ICK (12 ng/mL; equivalent to 1 ng/mL IL2) was added to media along with T cells and target cells at the beginning of 24 hours co-culture. For positive controls, 20% Triton x-100 (Sigma-Aldrich, X100-100ML) in PBS was added to wells with only target cells and incubated for 30 minutes at 37°C. Supernatants were removed and collected for measurement of IFNγ by ELISA. For quantitative analysis of GFP, culture media were replaced with fresh 200 µL of RPMI 1640 medium without phenol red and GFP fluorescence was read on a CLAROstar instrument. Mouse IFNγ ELISA (BioLegends, 430806) was performed per manufacturer’s directions. Supernatants were collected from each well of in vitro killing assay plate and diluted 1:5 for IFNγ ELISA.
Rabdosia rubescens Linn: green synthesis of gold nanoparticles and their anticancer effects against human lung cancer cells A549
Published in Artificial Cells, Nanomedicine, and Biotechnology, 2019
Xi Zhang, Zhenyue Tan, Kunjing Jia, Wenzhi Zhang, Minyan Dang
Human Adenocarcinomic alveolar basal epithelial (A549) cells and Human Medical Research Council cell strain 5 (MRC-5) lung cancer cells were procured from the Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences (Shanghai, China) and ATCC, USA, respectively. The cells were cultured in Dulbecco’s modified Eagle medium (DMEM)/F12 medium throughout the experimental period. The medium was supplemented with 10% fetal bovine serum and 1% antibiotic-antimycotic (penicillin-streptomycin) solution was added to prevent cells from contamination. The cells were incubated in CO2 incubator maintained at 37 °C with 5% CO2 supply. The culture medium was replaced for every 48 h or if the medium colour changes to yellow. Upon reaching 80% confluency, the cells were subcultured using 0.25% trypsin along with 0.02% ethyldiaminetetaacetic acid (Trypsin EDTA).