The pH Paradox in Reperfusion Injury to Heart Cells
John J. Lemasters, Constance Oliver in Cell Biology of Trauma, 2020
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.
Order Sepolyvirales
Paul Pumpens, Peter Pushko, Philippe Le Mercier in Virus-Like Particles, 2022
Goldmann et al. (2000) were the first to use the JCPyV VLPs as putative carriers for pharmaceutical substances of low molecular mass. Thus, propidium iodide (PI) was packaged into the VLP as a fluorescent marker, and the PI-containing VLPs were followed directly by flow cytometry. Qu et al. (2004) conjugated the E. coli-produced JCPyV VLPs with fluorescein isothiocyanate (FITC) and packaged the fluorescent dye Cy3 into the FITC-labeled VLPs. Ohtake et al. (2008) improved the uptake of the FITC-labeled VLPs by display of sialic acid. Ohtake et al. (2010) went to the direct incorporation of the visualizing proteins into the JCPyV VLPs, fused GFP to the N terminus of VP2, and coexpressed the fusion together with VP1 in E. coli. The expressed VP1 and GFP-VP2 associated with each other and formed the GFP-incorporated VLPs inside E. coli. Furthermore, the GFP-VP2/VP1 VLPs were provided with the His6 tag that was added N-terminally to the GFP-VP2, and the resultant VLPs were packed with nitrilotriacetic acid-sulforhodamine (NTA-SR), which contained both a His6-tag-targeting NTA segment and a fluorescent sulforhodamine SR segment. The cellular uptake and release of encapsulated NTA-SR was examined in NIH3T3 cells, where encapsulation of the NTA-SR markedly enhanced the cellular uptake compared to the NTA-SR alone (Ohtake et al. 2010). In fact, this was the first report of small molecule encapsulation within VLPs using specific peptide-tag interaction and of the controlled release in an environment-responsive manner.
Associated Methods
Lars-Inge Larsson in 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
Physico-chemical and antimicrobial properties and the shelf life of experimental endodontic sealers containing metal methacrylates
Published in Biofouling, 2020
Victoria Burmann da Silva Guimarães, Andressa da Silva Barboza, Carlos Enrique Cuevas-Suárez, Tiago Collares, Thaís Larré Oliveira, Anderson Schwingel Ribeiro, Meibel Teixeira Lisboa, Fernanda Geraldo Pappen, Rafael Guerra Lund
Biofilm viability analysis was performed using the SYTO 9 propidium iodide (Live/dead Bacligth Kit, Invitrogen, Eugene, OR, USA) technique. SYTO 9 is a green fluorescent stain that labels living and dead microorganisms. Propidium iodide is a red fluorescent nucleic acid patch that only penetrates cells with damaged membranes (dead cells). First, the samples were cleaned with 2ml of saline solution, and then 0.25μl of fluorescent stain was placed on the biofilm. A TCS SP8 CLSM (Leica Microsystems, Nussloch GmbH 2019, USA) was used to visualize the samples. The biofilm was evaluated at 100x magnification randomly. Then, five confocal stacks (10µm-20 slices of 0.5µm) of different random areas of each sample were obtained using a×10 lens and with the format of 512 × 512 pixels. The investigator was blinded to the experimental groups. All images were analyzed using LAS X 3D Analysis (Leica Microsystems, Nussloch GmbH 2019, USA) software for total biovolume (μm3), the total number of live cells (green) and the percentage of live cells.
Discovery of necrosis avidity of rhein and its applications in necrosis imaging
Published in Journal of Drug Targeting, 2020
Dongjian Zhang, Qiaomei Jin, Yicheng Ni, Jian Zhang
Furthermore, it was found that the uptakes of radiolabeled rhein derivatives in necrotic tissues could be significantly reduced by rhein itself or its unlabelled rhein derivatives, suggesting that the uptakes of rhein and its derivatives in necrotic tissues were target-specific [45,46,76]. Propidium iodide as a membrane-impermeable DNA staining reagent, which releases red fluorescence after insertion of double-stranded DNA, has long been used as a marker for necrosis in flow cytometry. In our studies, in vivo propidium iodide staining was used to verify DNA exposure in necrotic tissues [46,76]. It was demonstrated that a significantly higher red fluorescence intensity released by propidium iodide was observed in necrotic tissues than in normal tissues and that propidium iodide was mainly located in the nuclei of necrotic cells, which indicated that the loss of plasma membrane integrity of necrotic cells allowed the free access of propidium iodide to intracellular DNA, or in other words, the intracellular DNA became exposed in necrotic tissues [46,76]. In summary, we concluded that the necrosis avidity of rhein and its derivatives was to a large extent resulted from their binding to exposed DNA in necrotic tissues.
Development of Lactobacillus kimchicus DCY51T-mediated gold nanoparticles for delivery of ginsenoside compound K: in vitro photothermal effects and apoptosis detection in cancer cells
Published in Artificial Cells, Nanomedicine, and Biotechnology, 2019
Yeon-Ju Kim, Haribalan Perumalsamy, Josua Markus, Sri Renukadevi Balusamy, Chao Wang, Seong Ho Kang, Seungah Lee, Sang Yong Park, Sung Kim, Verónica Castro-Aceituno, Seung Hyun Kim, Deok Chun Yang
However, the number of apoptotic cells in the DCY51T-AuCKNps groups was significantly higher than those in the DCY51T-AuNps groups at the same concentration (5 µg/mL). Moreover, cells incubated with DCY51T-AuCKNps (both 1 and 5 µg/mL) were shown to demonstrate higher apoptosis in stomach, lung and colon adenocarcinoma cells than RAW264.7 cells. Increasing the concentration of DCY51T-AuCKNps from 1 to 5 µg/mL also resulted in higher apoptotic cells in cancer cell lines. We have also established increasing cell apoptotic mediated cell death in stomach cancer cell line (AGS) through a dose-dependent manner by staining the AGS cell lines with propidium iodide (Supplementary Figure S1). The cells treated with DCY51T-AuCKNps and laser treatment caused cell damages which led to dead cells. Staining the dead cells with propidium iodide resulted in the appearance of red fluorescence.
Related Knowledge Centers
- Cell Cycle Analysis
- Necrosis
- Nucleic Acid
- Quantum Yield
- Rna
- Apoptosis
- Flow Cytometry
- Staining
- Cell
- Viability Assay