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Structural Determination of the Polycystin-2 Channel by Electron Cryo-Microscopy
Published in Jinghua Hu, Yong Yu, Polycystic Kidney Disease, 2019
The BacMam protocol includes four steps: subcloning into a BacMam compatible vector, recombinant bacmids production, baculovirus generation, and protein expression in mammalian cell culture (Figure 2.1). The target gene is first subcloned into a BacMam compatible vector with the transposon element for bacmid generation in DH10Bac E. coli cells. Positive clones are selected using blue–white screening, and extracted bacmids are introduced into an insect cell host to produce baculoviruses for protein expression in the mammalian host.
Porphyromonas gingivalis W83 traffics via ICAM1 in microvascular endothelial cells and alters capillary organization in vivo
Published in Journal of Oral Microbiology, 2020
L. Reyes, H. Getachew, W.A. Dunn, A. Progulske-Fox
Colocalization of P. gingivalis with autophagosomes, early endosomes, or late endosomes was achieved by transduction with fluorescently tagged vectors inoculated at an MOI of 10 forty-eight hours before infection. Autophagosomes were tagged with green fluorescent protein (GFP)-light chain three (LC3) packaged in an adenovirus expression system (Welgen, Inc, Worcester, MA, USA). Both Rab5 (early endosome) and Rab7 (late endosome) were tagged with red fluorescent protein (RFP) packaged in a baculovirus system (CellLight® BacMam 2.0, Life Technologies, Grand Island, NY). Caveolae were labeled with a rabbit polyclonal antibody to caveolin-1 used at a dilution of 1:200 (catalog # ab18199, Abcam, Cambridge, MA). Late phagosomes/lysosomes were labeled with rabbit polyclonal antibody to LAMP1 used at a 1:200 dilution (catalog # SC-5570, Santa Cruz Biotechnology, Inc, Dallas, TX). Goat anti-mouse or anti-rabbit antibodies labeled with ALEXA 594 (absorption 590, emission 617) or ALEXA 647 (absorption 650, emission 665) (Life Technologies, Grand Island, NY) were used as secondary antibodies.
Effect of the flame retardant tris (1,3-dichloro-2-propyl) phosphate (TDCPP) on Na+-K+-ATPase and Cl− transport in HeLa cells
Published in Toxicology Mechanisms and Methods, 2018
Simona Latronico, Maria Elena Giordano, Emanuela Urso, Maria Giulia Lionetto, Trifone Schettino
Cellular Cl− fluxes were measured using Premo Halide Sensor (Life Technologies, Carlsbad, CA) according to the manufacturer instructions. Briefly, HeLa cells were seeded in plates at 70%–80% confluency with DMEM (10% FBS) for ∼24 h at 37 °C and 5% CO2 before proceeding with the transduction using BacMam delivery technology (Invitrogen, Carlsbad, CA). Cells were incubated with 1 ml D-PBS without Ca2+/Mg2+ containing transduction mixture (Invitrogen Cat. no. P10229; 1:1) at room temperature (20–25 °C) for 2–4 h with gentle rocking. Then, the cells were washed twice with PBS. Cells were incubated for 2 h with BacMam enchancer (1:1000). Then, the medium was discarded and the cells were washed twice with PBS. The incubation was continued for additional 18 h.
IFN-γ-induced ER stress impairs autophagy and triggers apoptosis in lung cancer cells
Published in OncoImmunology, 2021
Can Fang, Tao Weng, Shaojie Hu, Zhiwei Yuan, Hui Xiong, Bing Huang, Yixin Cai, Lequn Li, Xiangning Fu
The Premo™ Autophagy Tandem Sensor RFP-GFP-LC3B Kit was obtained from Invitrogen. The cells were transfected with BacMam reagent according to the manufacturer’s instructions. Subsequently, the cells were treated with Hank’s balanced salt solution (HBSS) and chloroquine (Enzo Life Sciences, Farmingdale, NY, USA). For the IFN-γ treatment group, the cells were pretreated with IFN-γ for 48 h and then transfected with BacMam reagents. After treatment, the cells were fixed with 4% paraformaldehyde and stained with DAPI. We quantified the numbers of red and yellow puncta using the Analyze Particle plugin for FIJI. A hue of 0–20 was counted as red puncta. A hue of 20–45 was counted as yellow puncta. We analyzed at least 20 randomly selected cells from each culture condition.