Injection Force Effects on Propidium Iodide Uptake in Nanoinjected HeLa Cells

2015 ◽  
Author(s):  
Tyler E. Lewis ◽  
Brian D. Jensen ◽  
Sandra H. Burnett
Keyword(s):  
Propidium Iodide ◽  
Dna Sequences ◽  
Hela Cells ◽  
Medical Community ◽  
Maximum Efficiency ◽  
Iodide Uptake ◽  
Advantages And Disadvantages ◽  
Area Of Interest ◽  
Slow Rise ◽  
Injection Force

The ability to deliver DNA sequences and molecular loads into the nucleus of a cell is a large area of interest in the medical community, and has numerous applications including gene therapy. Different methods of gene delivery each have their own advantages and disadvantages. Experimentation was performed using a new technique called nanoinjection to determine how force affects the efficiency and viability of the injection process. Nanoinjection, which utilizes an array of millions of solid microlances made on a 2 cm square silicon chip to inject cells, allows us to inject hundreds of thousands of cells at a time with high efficiencies. The solid nature of the lances allows them to be smaller, increasing cell viability. HeLa cells were used for testing in conjunction with an injection device that allowed for precise control of the injection force. Propidium iodide (PI), a dye that fluoresces when bound to DNA and doesn’t penetrate the membrane of living cells, was injected to monitor the efficiencies of transporting molecular loads across the cell membrane. The data was normalized to the controls for both efficiency (PI uptake) and viability to make it easier to see and determine trends. Results indicate a slow rise in injection efficiency from 0 to 1.8 Newtons of force where it reaches a maximum efficiency of 4.11 when normalized to the PI uptake of the positive controls. The data then levels, averaging an efficiency of approximately 3.1. The slow rise is likely due to more of the cells being punctured as the force increases until most have been punctured and the efficiency levels off. Viability had no clear trend, only varying between 0.97 and 1.04 when normalized to the viability of the negative controls.

Injection of Propidium Iodide into HeLa Cells Using a Silicon Nanoinjection Lance Array

2014 ◽  
Vol 5 (2) ◽  
Author(s):  
Zachary K. Lindstrom ◽  
Steven J. Brewer ◽  
Melanie A. Ferguson ◽  
Sandra H. Burnett ◽  
Brian D. Jensen
Keyword(s):  
Gene Therapy ◽  
Nucleic Acids ◽  
Propidium Iodide ◽  
Hela Cells ◽  
Human Cells ◽  
Advantages And Disadvantages ◽  
Culture Cells ◽  
High Viability ◽  
Promising Area ◽  
Hela Cell Culture

Delivering foreign molecules into human cells is a wide and ongoing area of research. Gene therapy, or delivering nucleic acids into cells via nonviral or viral pathways, is an especially promising area for pharmaceutics. All gene therapy methods have their respective advantages and disadvantages, including limited delivery efficiency and low viability. We present an electromechanical method for delivering foreign molecules into human cells. Nanoinjection, or delivering molecules into cells using a solid lance, has proven to be highly efficient while maintaining high viability levels. This paper describes an array of solid silicon microlances that was tested to determine efficiency and viability when nanoinjecting tens of thousands of HeLa cells simultaneously. Propidium iodide (PI), a dye that fluoresces when bound to nucleic acids and does not fluoresce when unbound, was delivered into cells using the lance array. Results show that the lance array delivers PI into up to 78% of a nanoinjected HeLa cell culture, while maintaining 78–91% viability. With these results, we submit the nanoinjection method using a silicon lance array as another promising particle delivery method for mammalian culture cells.

Design and Testing of an Automated Multi-Cell Nanoinjection System

2014 ◽  
Author(s):  
Zachary K. Lindstrom ◽  
Nicholas Gregory ◽  
Brian D. Jensen
Keyword(s):  
Propidium Iodide ◽  
Iodide Uptake ◽  
Membrane Pores ◽  
Culture Cells ◽  
The Press ◽  
Optimal Force ◽  
Experimental Variability ◽  
Cell Sample ◽  
Injection Force ◽  
Force Range

An automated nanoinjection system has been developed and tested for the delivery of propidium iodide into culture cells. Nanoinjection is the process by which molecules are delivered into living cells using a solid needle. Propidium iodide, a dye that fluoresces when bound to nucleic acids, was used as the injection molecule to monitor nanoinjection efficiency. The nanoinjection system uses a programmable microcontroller to manipulate a linear actuator, which presses a silicon lance array into thousands of living culture cells simultaneously. The lances penetrate cell membranes, allowing dye molecules to enter the cell through membrane pores opened by lances. The system was developed to apply the same injection force to each cell sample at the press of a button, eliminating any experimental variability in data due to the operator. Tests were performed at a dye concentration of 0.04 mg/mL for all experiments. Several forces were tested to determine the optimal nanoinjection force needed for maximum dye delivery. We found the optimal force range to be 8.8–14.7 N. The average PI uptake into cells at a force of 8.8 N and 14.7 N is 57.6±7.7% and 60.3±6.6%, respectively. Previous studies with a manual injection force have shown average propidium iodide uptake to be 60.4±18.0%. High cell viability is maintained with the automated nanoinjection system. At all forces applied in this experiment, an average of 78% or greater viability was observed. With the data gathered in this experiment, we conclude that the automated nanoinjection system eliminates much of the uptake efficiency variability inherent to nanoinjections performed with a manual injection force.

Catfish egg lectin affects influx and efflux rates of sunitinib in human cervical carcinoma HeLa cells

Glycobiology ◽  
2020 ◽  
Vol 30 (10) ◽  
pp. 802-816
Author(s):  
Shigeki Sugawara ◽  
Madoka Takayanagi ◽  
Shota Honda ◽  
Takeo Tatsuta ◽  
Yuki Fujii ◽  
...  
Keyword(s):  
Propidium Iodide ◽  
Hela Cells ◽  
Cytotoxic Effect ◽  
Kinase Inhibitor ◽  
Iodide Uptake ◽  
Treatment Protocols ◽  
Raji Cells ◽  
Silurus Asotus ◽  
New Treatment ◽  
Multitargeted Tyrosine Kinase Inhibitor

Abstract New treatment protocols are aiming to reduce the dose of the multitargeted tyrosine kinase inhibitor sunitinib, as sunitinib elicits many adverse effects depending on its dosage. Silurus asotus egg lectin (SAL) has been reported to enhance the incorporation of propidium iodide as well as doxorubicin into Burkitt’s lymphoma Raji cells through binding to globotriaosylceramide (Gb3) on the cell surface. The objective of this study was to examine whether SAL enhances the cytotoxic effect of sunitinib in Gb3-expressing HeLa cells. Although the treatment with SAL delayed the cell growth and enhanced the propidium iodide uptake, cell death accompanied by membrane collapse was not observed. The viability of sunitinib-treated HeLa cells was significantly reduced when the treatment occurred in combination with SAL compared to their separate usage. Sunitinib uptake significantly increased for 30 min in SAL-treated cells, and this increment was almost completely abolished by the addition of L-rhamnose, a hapten sugar of SAL, but not by D-glucose. After removal of SU from the medium, the intracellular sunitinib level in SAL-treated cells was higher than in untreated cells for 24 h, which was not observed in Gb3-deficient HeLa cells. Furthermore, we observed that SAL promoted the formation of lysosome-like structures, which are LAMP1 positive but not acidic in HeLa cells, which can trap sunitinib. Interestingly, SAL-induced vacuolation in HeLa cells was not observed in another Gb3 positive Raji cells. Our findings suggest that SAL/Gb3 interaction promoted sunitinib uptake and suppressed sunitinib excretion and that sunitinib efficiently exerted cytotoxicity against HeLa cells.

scholarly journals Specific growth inhibitory sequences in genomic DNA from quiescent human embryo fibroblasts.

1987 ◽  
Vol 7 (5) ◽  
pp. 1894-1899 ◽  
Author(s):  
R Padmanabhan ◽  
T H Howard ◽  
B H Howard
Keyword(s):  
Dna Sequences ◽  
Hela Cells ◽  
Human Embryo ◽  
Inhibitory Activity ◽  
Genomic Dna ◽  
Molecular Mechanisms ◽  
Growth Inhibitory ◽  
Growth Inhibitory Activity ◽  
Embryo Fibroblasts

We used HeLa cells as recipients in a gene transfer assay to characterize DNA sequences that negatively regulate mammalian cell growth. In this assay, genomic DNA from quiescent human embryo fibroblasts was more inhibitory for HeLa replication than was DNA from either Escherichia coli or HeLa cells. Surprisingly, growth inhibitory activity depended on the growth state of the cells from which genomic DNA was prepared; it was strongest in DNA prepared from serum-deprived, quiescent embryo fibroblasts. This latter observation implies a role for DNA modification(s) in regulating the activity of the inhibitory sequences detected in our assay. The level of the observed growth inhibitory activity was sometimes high, suggesting that the relevant sequences may be abundantly represented in the mammalian genome. We speculate that these findings may provide new insights into the molecular mechanisms involved in cellular quiescence and in vitro senescence.

scholarly journals Expression of amplified DNA sequences for ornithine transcarbamylase in HeLa cells: arginine residues may be required for mitochondrial import of enzyme precursor.

1985 ◽  
Vol 100 (5) ◽  
pp. 1515-1521 ◽  
Author(s):  
A L Horwich ◽  
W A Fenton ◽  
F A Firgaira ◽  
J E Fox ◽  
D Kolansky ◽  
...  
Keyword(s):  
Dna Sequences ◽  
Hela Cells ◽  
Regulatory Elements ◽  
Ornithine Transcarbamylase ◽  
Mitochondrial Localization ◽  
Mitochondrial Import ◽  
Cdna Sequences ◽  
Cell Extracts ◽  
Arginine Residues ◽  
Sequence Encoding

Expression of ornithine transcarbamylase (OTC), a nuclear-coded mitochondrial enzyme, was programmed in HeLa cells by the use of a strategy of gene co-amplification. HeLa cells, ordinarily devoid of OTC activity, were transfected with a plasmid containing viral regulatory elements joined with two cDNA sequences, one encoding the human OTC precursor and a second encoding a mutant mouse dihydrofolate reductase. After transfection and selection in increasing concentrations of methotrexate, several hundred copies per cell of the sequence encoding OTC were detected by blot analysis. Immunoprecipitation of extracts of radiolabeled cells with anti-OTC antiserum revealed newly synthesized mature OTC subunits. Furthermore, OTC enzymatic activity in cell extracts was comparable to that of control human liver, and mitochondrial localization of OTC was demonstrated by immunofluorescence. When we incubated transfected HeLa cells with dinitrophenol, a known inhibitor of mitochondrial import, the only form of newly synthesized OTC detected was the precursor. We estimated the rate of import of precursor by performing an inhibitor-free chase; precursor was converted to mature subunit with a half-life of less than two minutes. When a HeLa transformant was incubated with the arginine analogue canavanine, the major form of newly synthesized OTC detected was a species migrating slightly more slowly than the normal precursor; little mature-sized subunit was recovered. This indicates that substitution of the analogue for arginine in the OTC precursor interferes with mitochondrial import and processing. Thus, arginine residues in the OTC precursor--most likely the four residues contained in its NH2-terminal leader sequence--probably play an important role in mitochondrial import and/or processing.

scholarly journals Solar Cells: Current State and Development Prospects

2019 ◽  
Vol 62 (2) ◽  
pp. 105-123 ◽  
Author(s):  
I. I. Maronchuk ◽  
D. D. Sanikovich ◽  
V. I. Mironchuk
Keyword(s):  
Quantum Dots ◽  
Renewable Energy ◽  
Solar Cells ◽  
High Performance ◽  
Power Plants ◽  
National Laboratory ◽  
Thermal Power ◽  
Wave Radiation ◽  
Maximum Efficiency ◽  
Advantages And Disadvantages

The paper considers the main trends in the development of the world market of solar photovoltaics over the past few years. It is shown that the industry is a very rapidly evolving one among the branches of renewable energy and modern industries as a whole. It is obvious that the prime cost of the of solar energy being produced is rapidly approaching the price of electricity generated by traditional methods at nuclear power plants and thermal power plants. The aspects of the development of the efficiency of modern research solar cells made of various materials using innovative technological solutions based on the data provided by the National Laboratory for Renewable Energy (NREL, USA) in 2017 are noted. For the convenience of analysis, the research solar cells are divided into four technological groups. The advantages and disadvantages of solar cells, including the specific features of their production and prospects for development are considered separately for each group; the maximum efficiency for the year 2017 is estimated. A possible alternative to the future development of modern high-performance single-transition solar cells is the use of fundamentally new materials based on nanoheteroepitaxial structures with quantum dots. The possibilities of absorption (processing) by such structures of both short-wave radiation and long-wave part of the solar radiation spectrum for the purpose of generation of electric energy by increasing the efficiency of solar cells on their basis have been demonstrated. The optimal materials for their production and the principles of action of high-performance solar cells on their basis have been considered. The prospects of manufacturing nanoheteroepitaxial structures with quantum dots by liquid-phase epitaxy with pulse cooling of the substrate have been substantiated.

scholarly journals Study on Reconstruction Techniques of the Nipple-Areola Complex

2019 ◽  
Vol 70 (4) ◽  
pp. 1234-1238
Author(s):  
Silviu Adrian Marinescu ◽  
Catalin Gheorghe Bejinariu ◽  
Andreea Badeana ◽  
Sabina Tanasescu-Ciuvica ◽  
Dan Mircea Enescu
Keyword(s):  
Surgical Techniques ◽  
Medical Community ◽  
Duration Of Treatment ◽  
Basic Principles ◽  
Oncological Treatment ◽  
Nipple Areola Complex ◽  
Advantages And Disadvantages ◽  
Cancer Breast ◽  
Areola Complex

Increasing the incidence of breast cancer worldwide is an alarming factor for the medical community and also an additional reason for accountability. Oncological treatment, followed by subsequent reconstructive stages, are traumatic events that without a doubt exert their adverse effects on the quality of life of these patients. The reconstruction of the nipple-areola complex represents the final element in the treatment of patients who have suffered from cancer breast and must follow basic principles such as: reproducibility, efficacy, short duration of treatment and low rate of complications. The present material presents the various surgical techniques dedicated to the reconstruction of the nipple-areola complex, presenting their advantages and disadvantages based on a 5-year study. The use of single-stage techniques associated with medical tattooing were the main factor in increasing the satisfaction of the patients included in the research.

scholarly journals Genomic methods in profiling DNA accessibility and factor localization

2019 ◽  
Vol 28 (1) ◽  
pp. 69-85 ◽  
Author(s):  
David C. Klein ◽  
Sarah J. Hainer
Keyword(s):  
Gene Expression ◽  
Dna Sequences ◽  
Protein Localization ◽  
Regulation Of Gene Expression ◽  
Regulatory Regions ◽  
Histone Proteins ◽  
Putative Gene ◽  
Factor Binding ◽  
Advantages And Disadvantages ◽  
Dna Accessibility

AbstractRecent advancements in next-generation sequencing technologies and accompanying reductions in cost have led to an explosion of techniques to examine DNA accessibility and protein localization on chromatin genome-wide. Generally, accessible regions of chromatin are permissive for factor binding and are therefore hotspots for regulation of gene expression; conversely, genomic regions that are highly occupied by histone proteins are not permissive for factor binding and are less likely to be active regulatory regions. Identifying regions of differential accessibility can be useful to uncover putative gene regulatory regions, such as enhancers, promoters, and insulators. In addition, DNA-binding proteins, such as transcription factors that preferentially bind certain DNA sequences and histone proteins that form the core of the nucleosome, play essential roles in all DNA-templated processes. Determining the genomic localization of chromatin-bound proteins is therefore essential in determining functional roles, sequence motifs important for factor binding, and regulatory networks controlling gene expression. In this review, we discuss techniques for determining DNA accessibility and nucleosome positioning (DNase-seq, FAIRE-seq, MNase-seq, and ATAC-seq) and techniques for detecting and functionally characterizing chromatin-bound proteins (ChIP-seq, DamID, and CUT&RUN). These methods have been optimized to varying degrees of resolution, specificity, and ease of use. Here, we outline some advantages and disadvantages of these techniques, their general protocols, and a brief discussion of their development. Together, these complimentary approaches have provided an unparalleled view of chromatin architecture and functional gene regulation.

The stress protein ERp57/GRP58 binds specific DNA sequences in HeLa cells

2006 ◽  
Vol 210 (2) ◽  
pp. 343-351 ◽  
Author(s):  
Silvia Chichiarelli ◽  
Anna Ferraro ◽  
Fabio Altieri ◽  
Margherita Eufemi ◽  
Sabina Coppari ◽  
...  
Keyword(s):  
Dna Sequences ◽  
Hela Cells ◽  
Stress Protein

scholarly journals Flow microfluorometric analysis of cellular DNA: Critical comparison of mithramycin and propidium iodide.

1980 ◽  
Vol 28 (10) ◽  
pp. 1125-1128 ◽  
Author(s):  
V T Hamilton ◽  
M C Habbersett ◽  
C J Herman
Keyword(s):  
Propidium Iodide ◽  
Hela Cells ◽  
Cell Populations ◽  
Lymphoma Cells ◽  
Fluorescent Intensity ◽  
Critical Comparison ◽  
Structural Differences ◽  
Chromatin Structural ◽  
Human Lymphoma ◽  
Cellular Dna

Mithramycin and propidium iodide were used to stain HeLa cells, human lymphoma cells, and phytohemagglutinin-stimulated lymphocytes for flow microfluorometric analysis of cellular DNA. The stains provided similar estimates for the proliferative fraction of the populations. However, significant differences in the relative fluorescent intensity were demonstrated in the three cell populations. Fluorescent intensity of HeLa and lymphoma cells stained with mithramycin was higher than matched propidium iodide-stained cells. Normal lymphocytes showed greater fluorescent intensity when stained with propidium iodide. Differences in the staining behavior of these two dyes may prove to be highly informative probes of chromatin structural differences.

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