China
Africa
Explore chapters and articles related to this topic
Intracellular Double Labeling of Substantia Nigra and Pedunculopontine Neurons in in vitro Slice Preparation
Published in Avital Schurr, Benjamin M. Rigor, BRAIN SLICES in BASIC and CLINICAL RESEARCH, 2020
As for the immunocytochemical procedure, the following modification was made in the case of Lucifer Yellow labeling. The procedure for the primary antibody is the same, but for the secondary antibody, biotinylated secondary antibody (1:250 dilution, Vector) with 0.1 M PBS containing 0.5% Triton™ X-100 is used. Slices are incubated in the secondary antibody for 3 h at room temperature. Then, they are rinsed in 0.1 M PBS three times (10 min each) and incubated in Texas Red avidin (1:200 dilution) in 0.1 M PBS for 2 h at room temperature. This procedure is more effective for immunofluorescent reactions than using the FITC conjugated secondary antibody mentioned above. Slices are then rinsed three times (10 min each) in 0.1 M PBS, after which they are mounted on gelatin-coated glass slides and coverslipped with PBS containing 50% glycerol and 2.5% DABCO. Now the slides can be examined under an epifluorescence microscope (Nikon). Double labeled neurons are visualized for yellow using a UV-1A filter for the LY-labeled neuron and red using a G-2A filter for transmitter identification.
Intercellular Communication in Three-Dimensional Culture
Published in Rolf Bjerkvig, Spheroid Culture in Cancer Research, 2017
Most investigations on intercellular communication have been performed with cells in monolayer cultures. It seems, therefore, essential to question whether these results obtained for the two-dimensional case are also valid in three-dimensional growth — the natural situation in animals. Many coupling-competent cells remain coupled over a wide range of culture conditions (e.g., cell density, pH, or temperature of medium, serum, etc.) when they grow as monolayer cultures. This coupling is best visualized by the injection of Lucifer yellow and its diffusion into neighboring cells. However, when dye spreading is not observed, this does not necessarily indicate an absence of intercellular communication.16 The complete closure of gap-junction channels can only be demonstrated with high-resolution electrophysiology, using the double whole-cell patch-clamp technique.47 This method can only be performed with a sufficient accuracy at isolated cell pairs; in multicell spheroids, complex junction paths obscure these measurements. A total absence of gap junctions in a cell line must be ascertained with molecular techniques by demonstrating the lack of gap-junction RNA or protein.
Effect of functional excipients on the dissolution and membrane permeation of furosemide formulated into multiple-unit pellet system (MUPS) tablets
Published in Pharmaceutical Development and Technology, 2022
C. J. Van der Merwe, J. D. Steyn, J. H. Hamman, W. Pheiffer, H. Svitina, B. Peterson, J. H. Steenekamp
The membrane integrity of the two types of excised intestinal tissues mounted in the Sweetana-Grass diffusion apparatus was evaluated by screening for LY permeation. Lucifer yellow is a molecule used as a hydrophilic marker for determining membrane integrity for in vitro permeation models. During the membrane integrity study, a concentration of 50 µg/ml LY was added to the apical compartment together with the specific buffer suitable for the tissue evaluated. Pre-warmed Krebs-Ringer bicarbonate buffer was added to the basolateral compartment. The permeability study was conducted in a similar manner to the permeability studies done for the MUPS tablet test formulations. Samples withdrawn from the basolateral chamber were analysed using a Spectramax® Paradigm (serial number: 33270-1142) multi-mode detection platform plate reader. The excitation wavelength was set at 485 and emission wavelength at 530 nm. The integrity of the tissue evaluated was considered viable for permeability testing if the LY transport was equal to or less than 3% (Padmaja et al. 2004).
Inhibition of Gap Junction–Mediated Intercellular Communication by Poly(I:C) in Cultured Human Corneal Fibroblasts
Published in Current Eye Research, 2020
Hui Zheng, Ye Liu, Dan Xu, Pingping Liu, Xiuxia Yang, Bing Li, Zimu Cao, Yang Liu, Xiaoshuo Zheng
The scrape loading technique was used to evaluate GJIC activity as described previously23 but with some modifications. Cells were plated in 35-mm dishes and cultured to confluency. They were deprived of serum for 24 h and then incubated first for 6 h in the absence or presence of NAC (3 mM)24 or JNK inhibitor II (10 μM) and then for 24 h in the additional absence or presence of poly(I:C) (10 μg/ml). The cells were then washed with divalent ion–free PBS before exposure at room temperature to the same solution containing Lucifer yellow (0.5 mg/ml). The cells in each dish were subjected to three linear scrapes with a razor blade to allow dye entry and were then incubated for 60 min at 37°C, washed, fixed with 4% paraformaldehyde for 10 min at room temperature, and examined with a fluorescence microscope (Axioskop 50, Carl Zeiss). The spread of Lucifer yellow among the cells was determined by averaging the maximal distances at which dye fluorescence could be measured by ImageJ (NIH, Bethesda, MD). At least three measurements were obtained along each scrape line.
An in vitro FcRn- dependent transcytosis assay as a screening tool for predictive assessment of nonspecific clearance of antibody therapeutics in humans
Published in mAbs, 2019
Shan Chung, Van Nguyen, Yuwen Linda Lin, Julien Lafrance-Vanasse, Suzie J. Scales, Kevin Lin, Rong Deng, Kathi Williams, Gizette Sperinde, Juan Jenny Li, Kai Zheng, Siddharth Sukumaran, Devin Tesar, James A. Ernst, Saloumeh Fischer, Greg A. Lazar, Saileta Prabhu, An Song
Cells were seeded at a density of 1 × 105 cells/well in cell growth medium (DMEM High Glucose supplemented with 10% FBS, 100 units penicillin/streptomycin, and 5 µg/mL puromycin) in 96-well trans-well plates (Cat# CLS3381, Corning Costar, Corning, NY, USA), with 100 and 200 µL of medium in the inner and outer chambers, respectively. Cells were used for experiments on the second day post-plating. The medium in the inner chamber was removed and test molecules were added to a final concentration of 100 µg/mL (0.67 µM) and incubated for 24 h at 37°C. The functional integrity of filter-grown MDCK-hFcRn cells was monitored by measuring trans-epithelial electrical resistance (TEER). The TEER of monolayers before the assay typically ranged from 250 to 300 Ω*cm2, a characteristic range for polarized MDCK II cells.30 The barrier integrity (leakiness) of the monolayer during the assay was monitored by spiking Lucifer Yellow (Lucifer Yellow CH, dilithium salt; Cat# L0259, Sigma Aldrich) along with the test molecules in the inner chamber. Lucifer Yellow prepared in cell growth medium was added in the final 90 min of the 24-h assay incubation. Media from the outer chamber were collected and the amount of transcytosed molecules was determined by ELISA. The level of passive passage of Lucifer Yellow during the assay was calculated by dividing the florescent signal in samples from the outer chamber by that of the inner chamber. Transcytosis results from wells exhibiting greater than 0.1% of passive passage of Lucifer Yellow in the outer chamber were discarded.