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Associated Methods
Published in Lars-Inge Larsson, Immunocytochemistry: Theory and Practice, 2020
Initial attempts to combine HRP retrograde transport with indirect immunofluorescence were reported by Ljungdahl et al.69 Subsequently, different fluorescent retrograde axonal transport markers have been found to survive processing for immunofluorescence and have therefore been useful for double fluorescence labeling of identified neurons.47,104,105 Propidium iodide (excited at 530 to 560 nm, yielding orange fluorescence) and true blue (excited at 340 to 380 nm, yielding blue fluorescence) were found to be compatible with each other in double-tracing experiments and with indirect immunofluorescence, using FITC.104
The pH Paradox in Reperfusion Injury to Heart Cells
Published in John J. Lemasters, Constance Oliver, Cell Biology of Trauma, 2020
John J. Lemasters, John M. Bond, Ian S. Harper, Enrique Chacon, Hisayuki Ohata, Brian Herman, Wayne E. Cascio
Propidium iodide is a DNA-intercalating fluorophore that is impermeable to the plasma membrane of viable cells. When viability is lost, propidium iodide rapidly gains entrance to cells and binds to nuclei with an enhancement of its fluorescence. Using nuclear labeling with propidium to signify loss of cell viability, we evaluated the pH dependence of lethal injury to cultured rat neonatal cardiac myocytes during anoxic stress. Initially, cells were incubated in an aerobic Krebs-Ringer-HEPES (KRH) buffer at pH 7.4. Anoxia was then imposed by infusing submitochondrial particles, succinate, and 2-deoxyglucose in KRH buffer. Submitochondrial particles oxidizing succinate have high respiratory rates and consume oxygen until it is undetectable by oxygen electrodes. Moreover, the continuing presence of submitochondrial particles assured that the myocytes were not reoxygenated by oxygen back-diffusion during the course of the experiments. 2-Deoxyglucose blocked glycolytic ATP formation from glycogen or glucose carried over from the culture medium. Under these conditions of anoxia, spontaneous contractions ceased within a few minutes.
Senescent Cells as Drivers of Age-Related Diseases
Published in Shamim I. Ahmad, Aging: Exploring a Complex Phenomenon, 2017
Cielo Mae D. Marquez, Michael C. Velarde
Cell cycle analysis may also be performed using flow cytometry. Flow cytometric analysis uses DNA stains that bind proportionally to the amount of DNA present within the cell [37]. The more dyes taken up, the brighter the fluorescence. This helps to distinguish senescent cells (arrested at G1 or G2/M phases) versus non-senescent cells (quiescent at G0 or undergoing G1/S phase transitions) [27,28]. DNA-binding dyes which are frequently used include propidium iodide (PI), 4′,6-diamidino-2-phenylindole (DAPI), 7-aminoactinomycin-D (7-AAD), and Hoechst 33342 [38]. Generally, cells must be fixed and/or permeabilized to allow passage of the dyes, which is otherwise excluded by live cells. Common fixatives applied are alcohol and aldehyde which may be used in combination with a detergent or other permeabilizing reagents. Alcohols fix cells by dehydration and protein denaturation, while aldehydes fix cells by cross-linking proteins and other macromolecules [39].
Enteropathogenic Escherichia coli regulates host-cell mitochondrial morphology
Published in Gut Microbes, 2022
Jennifer Lising Roxas, Shylaja Ramamurthy, Katie Cocchi, Ilga Rutins, Anusha Harishankar, Al Agellon, John Scott Wilbur, Gresa Sylejmani, Gayatri Vedantam, V.K. Viswanathan
Hela cells were transiently transfected with FIS1 shRNA or Scramble shRNA and FACS-sorted as described above. At 72 hours post-transfection, sorted cells (5000 cells/well) were seeded to 96-well black-walled, clear bottom microplates. Seven days after initial transfection, cells were infected as mentioned above. Unattached bacteria were carefully removed at 1-hour post-infection. Fresh medium containing 1 μg/mL of propidium iodide (PI; Abcam, Cambridge, MA) was added to the cells. PI uptake was monitored for 8 hours, and fluorescence readings taken at 30-minute intervals using a microplate reader (Synergy HT; BioTek Instruments, Winooski, VT) equipped with a 530/25 nm excitation and 620/40 nm emission filters. Epithelial cells killed with 70% methanol were used as controls to estimate maximum PI uptake.
AAV-mediated gene transfer of a checkpoint inhibitor in combination with HER2-targeted CAR-NK cells as experimental therapy for glioblastoma
Published in OncoImmunology, 2022
M.I. Strecker, K. Wlotzka, F. Strassheimer, B. Roller, G. Ludmirski, S. König, J. Röder, C. Opitz, T. Alekseeva, J. Reul, L. Sevenich, T. Tonn, W.S. Wels, J.P. Steinbach, C.J. Buchholz, M.C. Burger
Expression of HER2 on the surface of GL261-HER2, Tu2449-HER2, Tu9648-HER2 and LN-319 cells was determined by flow cytometry using AlexaFluor® 647 anti-human HER2 antibody 24D2 (BioLegend, Fell, Germany). Presence of mutated EGFRvIII on the surface of LNT-229-EGFRvIII cells was analyzed using an AlexaFluor® 488 anti-EGFR mutant antibody DH8.3 (Novus Biologicals). CAR expression of anti-HER2.CAR/NK-92 cells was analyzed using a HER2-Fc fusion protein (R&D Systems, Wiesbaden-Nordenstadt, Germany) followed by APC-coupled goat anti-human antibody (Dianova, Hamburg, Germany). PD-L1 expression by murine and human cells was determined using APC-coupled anti-mouse or anti-human PD-L1 antibodies, respectively. Cell viability was analyzed by propidium iodide uptake as previously described.37 A FACSCanto II flow cytometer was used for flow cytometric analyses (BD Biosciences, Heidelberg, Germany), and data were evaluated using FlowJo™ software. For cell sorting, a FACSAria flow cytometer was used (BD Biosciences).
Dyhidro-β-agarofurans natural and synthetic as acetylcholinesterase and COX inhibitors: interaction with the peripheral anionic site (AChE-PAS), and anti-inflammatory potentials
Published in Journal of Enzyme Inhibition and Medicinal Chemistry, 2022
Julio Alarcón-Enos, Evelyn Muñoz-Núñez, Margarita Gutiérrez, Soledad Quiroz-Carreño, Edgar Pastene-Navarrete, Carlos Céspedes Acuña
The ability of compounds to competitively displace propidium iodide was evaluated by a fluorescence method43. To determine the degree of displacement (% displacement) of propidium iodide from the PAS of AChE, EeAChE (fnal concentration 7 µM) was incubated with the test compound at a concentration of 290 and 340 µM in 1 mM Tris-HCl bufer pH 8.0, 25 °C for 15 min. Then, propidium iodide solution (final concentration 8 µM) was added, the samples were incubated for 15 min and the fluorescence spectrum 530 nm (excitation) and 600 nm (emission) was taken. Donepezil was used as reference compound. The blank contained propidium iodide of the same concentration in 1 mM Tris-HCl buffer pH 8.0. The measurements were carried out in triplicate on a microplate reader Perkin Elmer VictorX2 (Perkin Elmer, Singapur).