scholarly journals Perturbing the Normal Level of SIDT1 Suppresses the Naked ASO Effect

2021 ◽  
Vol 2021 ◽  
pp. 1-9
Author(s):  
Masayuki Takahashi ◽  
Mineaki Seki ◽  
Masayuki Nashimoto ◽  
Tomohiro Kabuta
Keyword(s):  
Flow Cytometry ◽  
Cellular Uptake ◽  
Normal Level ◽  
Antisense Oligonucleotide ◽  
Intracellular Trafficking ◽  
Microscopic Analysis ◽  
Living Cells ◽  
Therapeutic Effects ◽  
Fluorescence Microscopic Analysis ◽  
Antisense Effect

Although antisense oligonucleotide (ASO) therapeutics can be taken up by living cells without carrier molecules, a large part of incorporated ASOs are trapped in the endosomes and do not exert therapeutic effects. To improve their therapeutic effects, it would be important to elucidate the mechanism of cellular uptake and intracellular trafficking of ASOs. In this study, we investigated how SIDT1 affects cellular uptake and intracellular trafficking of ASOs. Fluorescence microscopic analysis suggested that most of naked ASOs are trafficked to the lysosomes via the endosomes. The data obtained from flow cytometry and fluorescence microscopy together showed that although the SIDT1 level barely affects the total cellular uptake of ASOs, it appears to affect the intracellular trafficking of ASOs. We also showed that SIDT1 exists mainly in the endoplasmic reticulum and that perturbing the normal level of SIDT1 suppresses the antisense effect of the naked ASO targeting miR-16.

scholarly journals Frequency domain fluorescence microspectrometry: Application to cellular uptake and drug distribution

2010 ◽  
Vol 24 (3-4) ◽  
pp. 303-307 ◽  
Author(s):  
Petr Praus ◽  
Eva Kocišová ◽  
Peter Mojzeš ◽  
Josef Štepánek ◽  
Franck Sureau ◽  
...  
Keyword(s):  
Cellular Uptake ◽  
Decay Time ◽  
Spectral Region ◽  
High Frequency ◽  
Antisense Oligonucleotide ◽  
Drug Distribution ◽  
Image Intensifier ◽  
Living Cells ◽  
Time Data ◽  
Time Resolved

Time-resolved confocal microspectrofluorometry and fluorescence microimaging were used to monitor how the model antisense oligonucleotide is transported into 3T3 living cells and distributed inside them. Phosphorothioate analog of 15-mer oligothymidylate labeled by ATTO 425 was complexed with Zn(II) 5,10,15,20-tetrakis(4-N-methylpyridyl) porphyrin as an uptake-mediating agent. Homodyne phase-resolved technique based on a high frequency analog modulation of both exciting diode laser and detector image intensifier was used for time-resolved measurements. Decay-time data obtained within a broad range spectral region have provided unique information about the fate of both fluorophores inside the cell.

Intracellular trafficking and cytoplasmic streaming under abiotic stress conditions

2019 ◽  
Vol 6 (04) ◽  
Author(s):  
JESHIMA KHAN YASIN ◽  
ANIL KUMAR SINGH
Keyword(s):  
Plasma Membrane ◽  
Abiotic Stress ◽  
Confocal Microscopy ◽  
Fusion Protein ◽  
Intracellular Trafficking ◽  
Cytoplasmic Streaming ◽  
Living Cells ◽  
Stress Conditions ◽  
Vesicle Formation ◽  
External Stimuli

Cytoplasmic streaming is one among the vital activities of the living cells. In plants cytolplasmic streaming could clearly be seen in hypocotyls of growing seedlings. To observe cytoplsmic streaming and its correlated intracellular trafficking an investigation was conducted in legumes in comparison with GFP-AtRab75 and 35S::GFP:δTIP tonoplast fusion protein expressing arabidopsis lines. These seedlings were observed under confocal microscopy with different buffer incubation treatments and under different stress conditions. GFP expressing 35S::GFP:δTIP tonoplast lines were looking similar to the control lines and differ under stress conditions. Movement of cytoplasmic invaginations within the tonoplast and cytoplasmic sub vesicle or bulb budding during cytoplasmic streaming was observed in hypocotyls of At-GFP tonoplast plants. We found the cytoplasmic bulbs/ vesicles or sub vesicle formation from the plasma membrane. The streaming speed also depends on the incubation medium in which the specimen was incubated, indicating that the external stimuli as well as internal stimuli can alter the speed of streaming

scholarly journals In Vitro Cellular Uptake Studies of Self-Assembled Fluorinated Nanoparticles Labelled with Antibodies

Nanomaterials ◽  
2021 ◽  
Vol 11 (8) ◽  
pp. 1906
Author(s):  
Mona Atabakhshi-Kashi ◽  
Mónica Carril ◽  
Hossein Mahdavi ◽  
Wolfgang J. Parak ◽  
Carolina Carrillo-Carrion ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Cellular Uptake ◽  
Hydrophobic Interactions ◽  
Intrinsic Properties ◽  
Wide Range ◽  
Self Assembled ◽  
Uptake Studies ◽  
Targeting Ability ◽  
Fluorinated Nanoparticles

Nanoparticles (NPs) functionalized with antibodies (Abs) on their surface are used in a wide range of bioapplications. Whereas the attachment of antibodies to single NPs to trigger the internalization in cells via receptor-mediated endocytosis has been widely studied, the conjugation of antibodies to larger NP assemblies has been much less explored. Taking into account that NP assemblies may be advantageous for some specific applications, the possibility of incorporating targeting ligands is quite important. Herein, we performed the effective conjugation of antibodies onto a fluorescent NP assembly, which consisted of fluorinated Quantum Dots (QD) self-assembled through fluorine–fluorine hydrophobic interactions. Cellular uptake studies by confocal microscopy and flow cytometry revealed that the NP assembly underwent the same uptake procedure as individual NPs; that is, the antibodies retained their targeting ability once attached to the nanoassembly, and the NP assembly preserved its intrinsic properties (i.e., fluorescence in the case of QD nanoassembly).

scholarly journals Measurement of Separase Proteolytic Activity in Single Living Cells by a Fluorogenic Flow Cytometry Assay

PLoS ONE ◽  
2015 ◽  
Vol 10 (8) ◽  
pp. e0133769 ◽  
Author(s):  
Wiltrud Haaß ◽  
Helga Kleiner ◽  
Martin C. Müller ◽  
Wolf-Karsten Hofmann ◽  
Alice Fabarius ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Proteolytic Activity ◽  
Living Cells ◽  
Flow Cytometry Assay ◽  
Single Living Cells ◽  
Single Living

scholarly journals A Flow Cytometry-Based FRET Assay to Identify and Analyse Protein-Protein Interactions in Living Cells

PLoS ONE ◽  
2010 ◽  
Vol 5 (2) ◽  
pp. e9344 ◽  
Author(s):  
Carina Banning ◽  
Jörg Votteler ◽  
Dirk Hoffmann ◽  
Herwig Koppensteiner ◽  
Martin Warmer ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Protein Interactions ◽  
Living Cells ◽  
Protein Protein Interactions

Surface chemistry modification of silica nanoparticles alters the activation of monocytes

2021 ◽  
Author(s):  
Romina Mitarotonda ◽  
Martín Saraceno ◽  
Marcos Todone ◽  
Exequiel Giorgi ◽  
Emilio L Malchiodi ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Immune System ◽  
Cell Membrane ◽  
Cell Activation ◽  
Membrane Damage ◽  
Living Cells ◽  
Cell Membrane Damage ◽  
Study Materials ◽  
Therapeutic Molecules ◽  
Pro Inflammatory Cytokines

Aim: Nanoparticles (NPs) interaction with immune system is a growing topic of study. Materials & methods: Bare and amine grafted silica NPs effects on monocytes/macrophages cells were analyzed by flow cytometry, MTT test and LIVE/DEAD® viability/cytotoxicity assay. Results: Bare silica NPs inhibited proliferation and induced monocyte/macrophages activation (increasing CD40/CD80 expression besides pro-inflammatory cytokines and nitrite secretion). Furthermore, silica NPs increased cell membrane damage and reduced the number of living cells. In contrast, amine grafted silica NPs did not alter these parameters. Conclusion: Cell activation properties of bare silica NPs could be hindered after grafting with amine moieties. This strategy is useful to tune the immune system stimulation by NPs or to design NPs suitable to transport therapeutic molecules.

In Vitro Anti-Biofilm Activity of Hydrogen-Peroxide Generating Electrochemical Bandage Against Yeast Biofilms

2021 ◽  
Author(s):  
Yash S. Raval ◽  
Abdelrhman Mohamed ◽  
Jayawant N. Mandrekar ◽  
Cody Fisher ◽  
Kerryl E. Greenwood-Quaintance ◽  
...  
Keyword(s):  
Membrane Damage ◽  
Microscopic Analysis ◽  
Viable Cell ◽  
Wound Infections ◽  
Unique Challenge ◽  
Cell Membrane Damage ◽  
Cell Counts ◽  
Extensive Cell ◽  
Fluorescence Microscopic Analysis

Wound infections are caused by bacteria and/or fungi. The presence of fungal biofilms in wound beds presents a unique challenge, as fungal biofilms may be difficult to eradicate. The goal of this work was to assess the in vitro anti-biofilm activity of a H 2 O 2 -producing electrochemical bandage (e-bandage) against 15 yeast isolates representing commonly-encountered species. Time-dependent decreases in viable biofilm CFU counts of all isolates tested were observed, resulting in no visible colonies with 48 hours of exposure by plate culture. Fluorescence microscopic analysis showed extensive cell membrane damage of biofilm cells after e-bandage treatment. Reductions in intracellular ATP levels of yeast biofilm cells were recorded post e-bandage treatment. Our results suggest that exposure to H 2 O 2 -producing e-bandages reduce in vitro viable cell counts of yeast biofilms, making this a potential new topical treatment approach for fungal wound infections.

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