The Experts below are selected from a list of 7740 Experts worldwide ranked by ideXlab platform
Pavel A. Levkin - One of the best experts on this subject based on the ideXlab platform.
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Superhydrophilic–Superhydrophobic Patterned Surfaces as High-Density Cell Microarrays: Optimization of Reverse Transfection
Advanced healthcare materials, 2016Co-Authors: Erica Ueda, Wenqian Feng, Pavel A. LevkinAbstract:High-density microarrays can screen thousands of genetic and chemical probes at once in a miniaturized and parallelized manner, and thus are a cost-effective alternative to microwell plates. Here, high-density cell microarrays are fabricated by creating superhydrophilic-superhydrophobic micropatterns in thin, nanoporous polymer substrates such that the superhydrophobic barriers confine both aqueous solutions and adherent cells within each superhydrophilic microspot. The superhydrophobic barriers confine and prevent the mixing of larger droplet volumes, and also control the spreading of droplets independent of the volume, minimizing the variability that arises due to different liquid and surface properties. Using a novel liposomal Transfection reagent, ScreenFect A, the method of Reverse cell Transfection is optimized on the patterned substrates and several factors that affect Transfection efficiency and cytotoxicity are identified. Higher levels of Transfection are achieved on HOOC- versus NH2 -functionalized superhydrophilic spots, as well as when gelatin and fibronectin are added to the Transfection mixture, while minimizing the amount of Transfection reagent improves cell viability. Almost no diffusion of the printed Transfection mixtures to the neighboring microspots is detected. Thus, superhydrophilic-superhydrophobic patterned surfaces can be used as cell microarrays and for optimizing Reverse cell Transfection conditions before performing further cell screenings.
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superhydrophilic superhydrophobic patterned surfaces as high density cell microarrays optimization of Reverse Transfection
Advanced Healthcare Materials, 2016Co-Authors: Erica Ueda, Wenqian Feng, Pavel A. LevkinAbstract:High-density microarrays can screen thousands of genetic and chemical probes at once in a miniaturized and parallelized manner, and thus are a cost-effective alternative to microwell plates. Here, high-density cell microarrays are fabricated by creating superhydrophilic-superhydrophobic micropatterns in thin, nanoporous polymer substrates such that the superhydrophobic barriers confine both aqueous solutions and adherent cells within each superhydrophilic microspot. The superhydrophobic barriers confine and prevent the mixing of larger droplet volumes, and also control the spreading of droplets independent of the volume, minimizing the variability that arises due to different liquid and surface properties. Using a novel liposomal Transfection reagent, ScreenFect A, the method of Reverse cell Transfection is optimized on the patterned substrates and several factors that affect Transfection efficiency and cytotoxicity are identified. Higher levels of Transfection are achieved on HOOC- versus NH2 -functionalized superhydrophilic spots, as well as when gelatin and fibronectin are added to the Transfection mixture, while minimizing the amount of Transfection reagent improves cell viability. Almost no diffusion of the printed Transfection mixtures to the neighboring microspots is detected. Thus, superhydrophilic-superhydrophobic patterned surfaces can be used as cell microarrays and for optimizing Reverse cell Transfection conditions before performing further cell screenings.
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Droplet-microarray on superhydrophobic–superhydrophilic patterns for high-throughput live cell screenings
RSC Advances, 2016Co-Authors: Anna A. Popova, Konstantin Demir, Titus Genisius Hartanto, Eric Schmitt, Pavel A. LevkinAbstract:Cell-based high-content phenotypic screenings are widely used in fundamental research, pharmaceutical industry, and healthcare in order to simultaneously evaluate the effects of multiple compounds or gene overexpressions/knockdown on the phenotype of cells. Most screenings, particularly in the industrial sector, are performed using the microplate technology, which relies on high reagent and cell consumption, as well as on expensive liquid-handling robotics. Developing miniaturized screening platforms has been an important topic in the past decade. In this study, we demonstrate the applicability of the droplet-microarray platform based on superhydrophobic–superhydrophilic patterning for cell-based high-throughput screenings. We show the homogeneous seeding of cells and culturing of different adherent cell lines in individual droplets of different sizes. We demonstrate pipetting-free medium exchange, enabling cell culture in miniaturized droplet arrays for up to 5 days. We establish the methods of Reverse Transfection and Reverse drug screening in individual nanoliter-sized droplets by printing Transfection mixtures or drug molecules directly onto superhydrophilic spots prior to cell seeding.
J U N Miyake - One of the best experts on this subject based on the ideXlab platform.
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New methods for Reverse Transfection with siRNA from a solid surface.
Methods in molecular biology (Clifton N.J.), 2010Co-Authors: Satoshi Fujita, Masato Miyake, Kota Takano, Eiji Ota, Takuma Sano, Tomohiro Yoshikawa, J U N MiyakeAbstract:We describe two efficient and inexpensive methods for Reverse Transfection with siRNA from a solid surface. One method involves localized Reverse Transfection from spots on a glass slide, which is mainly useful for making "Transfection microarrays" (TMAs). The other involves Reverse Transfection in multiple wells of microtiter plates. Conditions for cell culture, preparation of reagents, and details of Reverse Transfection have been determined for several lines of cells, but we focus here on experiments with HeLa cells. In particular, we evaluated the efficiency of Transfection, the cytotoxic effects of Reverse Transfection, and the efficiency of gene "knockdown" by Transfection. We also performed phenotypic screening for a functional gene, during which cell viability was evaluated in terms of fluorescence from Calcein-AM. Our methods for Reverse Transfection with siRNA should be powerful tools that are useful for high-throughput analysis of functional genes.
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Reverse Transfection using gold nanoparticles.
Methods in molecular biology (Clifton N.J.), 2009Co-Authors: Shigeru Yamada, Eiichiro Uchimura, Masato Miyake, Satoshi Fujita, J U N MiyakeAbstract:Reverse Transfection from a solid surface has the potential to deliver genes into various types of cell and tissue more effectively than conventional methods of Transfection. We present a method for Reverse Transfection using a gold colloid (GC) as a nanoscaffold by generating nanoclusters of the DNA/reagent complex on a glass surface, which could then be used for the regulation of the particle size of the com- plex and delivery of DNA into nuclei. With this method, we have found that the conjugation of gold nanoparticles (20 nm in particle size) to the pEGFP-N1/Jet–PEI complex resulted in an increase in the intensity of fluorescence of enhanced green fluorescent protein (EGFP) (based on the efficiency of Transfection) from human mesenchymal stem cells (hMSCs), as compared with the control without GC. In this manner, we constructed a method for Reverse Transfection using GC to deliver genes into the cells effectively.
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Highly efficient Reverse Transfection with siRNA in multiple wells of microtiter plates
Journal of bioscience and bioengineering, 2007Co-Authors: Satoshi Fujita, Masato Miyake, Kota Takano, Eiji Ota, Chie Sasaki, J U N MiyakeAbstract:We have developed an efficient and inexpensive method of Reverse Transfection from the solid phase to suppress genes with siRNA. The method enabled the realization of (i) a high efficiency of Transfection; (ii) Transfection of various types of cell; (iii) a high efficiency of gene knockdown by siRNA; (iv) a low toxicity to cells; and (v) a long-term stabilization (more than 210 d) of attached Transfection mixture including siRNA in multiple wells. Although array-based Reverse Transfection has advantages in terms of miniaturization, the method has the advantage of enabling the inclusion of various soluble factors, such as humoral factors, drugs and ligands that affect gene expression, because the liquid phase is partitioned within the individual wells of each microtiter plate. Our method of Reverse Transfection with siRNA in multiple wells is a powerful and high-throughput tool for the analysis of signaling pathways.
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Reverse Transfection using antibodies against a cell surface antigen in mammalian adherent cell lines
Journal of bioscience and bioengineering, 2007Co-Authors: Eiichiro Uchimura, Masato Miyake, Shigeru Yamada, Satoshi Fujita, Takashi Nomura, Kayo Matsumoto, J U N MiyakeAbstract:Reverse Transfection from a solid surface has the potential to deliver genes to various cells more efficiently than conventional methods. However, the effective gene delivery from a solid surface requires an optimized extracellular matrix (ECM) for the coating of glass slides, dependent on the nature of the cells. In a search for an appropriate substrate for the universal application to multiple types of cell, we focused on cell surface antigens and examined the effects of antibodies raised against them on gene transfer from an antibody-coated surface. We found that a coating of CD29-specific antibody allowed the most effective delivery of genes by Reverse Transfection in every type of cell that we examined. Our results suggest that Reverse Transfection with antibodies against CD29 might provide a universal tool for gene delivery and cell array-based analyses.
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Method for Reverse Transfection using gold colloid as a nano-scaffold.
Journal of bioscience and bioengineering, 2007Co-Authors: Eiichiro Uchimura, Masato Miyake, Shigeru Yamada, Satoshi Fujita, Lorenz Uebersax, J U N MiyakeAbstract:DNA microarray of non-viral Reverse Transfection in cell engineering allows drastic downsizing of large-scale functional screening of genes and siRNAs. However the control of localizability and efficiency of the microarray is still considered as a critical barrier in practical use. One of the major breakthrough to increase the Transfection efficiency may be control in the condition of DNA/Transfection reagent complex on the microarray surface. In this paper, we showed that negatively charged gold colloid (GC) is successfully used to control the DNA/reagent complex on a glass surface. The conjugation of gold nanoparticles (20 nm in diameter) to the pEGFP-N1/Jet-PEI complex resulted in a more than 2.5-fold increase in the intensity of fluorescence of enhanced green fluorescent protein (EGFP) (based on the efficiency of Transfection) from human mesenchymal stem cells (hMSCs), as compared to the control without GC. Our method for Reverse Transfection should be useful not only for cell array-based analyses but also as a novel gene-delivery method for gene therapy in regenerative medicine.
Rainer Pepperkok - One of the best experts on this subject based on the ideXlab platform.
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Work flow for multiplexing siRNA assays by solid-phase Reverse Transfection in multiwell plates.
Journal of biomolecular screening, 2008Co-Authors: Holger Erfle, Beate Neumann, Phill Rogers, Jutta Bulkescher, Jan Ellenberg, Rainer PepperkokAbstract:Solid-phase Reverse Transfection on cell microarrays is a high-throughput method for the parallel Transfection of mammalian cells. However, the cells transfected in this way have been restricted so far to microscopy-based analyses. Analysis methods such as Reverse transcriptase–polymerase chain reaction (RT-PCR) and access to higher cell numbers for statistical reasons in microscopy-based assays are not possible with solid-phase Reverse Transfection on cell microarrays. We have developed a quick and reliable protocol for automated solid-phase Reverse Transfection of human cells with siRNAs in multiwell plates complementing solid-phase Reverse Transfection on cell microarrays. The method retains all advantages of solid-phase Reverse Transfection such as long-term storage capacity after fabrication, reduced cytotoxicity, and reduced cost per screen compared with liquid-phase Transfection in multiwell plates. The protocol has been tested for the RNAi-mediated knockdown of several genes in different cell lines including U20S, RPE1, A549, and HeLa cells. We show that even 3 months after production of the “ready to transfect” multiwell plates, there is no reduction in their Transfection efficiency as assessed by RT-PCR and nuclear phenotyping by fluorescence microscopy. We conclude that solidphase Reverse Transfection in multiwell plates is a cost-efficient and flexible tool for multiplexing cellular assays. (Journal of Biomolecular Screening. 2008:575-580)
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Reverse Transfection on cell arrays for high content screening microscopy.
Nature protocols, 2007Co-Authors: Holger Erfle, Beate Neumann, Phill Rogers, Jan Ellenberg, Urban Liebel, Michael Held, Thomas Walter, Rainer PepperkokAbstract:Here, we describe a robust protocol for the Reverse Transfection of cells on small interfering (siRNA) arrays, which, in combination with multi-channel immunofluorescence or time-lapse microscopy, is suitable for genome-wide RNA interference (RNAi) screens in intact human cells. The automatic production of 48 'Transfection ready' siRNA arrays, each containing 384 samples, takes in total 7 h. Pre-fabricated siRNA arrays can be used without loss of Transfection efficiency at least up to 15 months after printing. Different human cell lines that have been successfully transfected using the protocol are presented here. The present protocol has been applied to two genome-wide siRNA screens addressing mitosis and constitutive protein secretion.
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Arrays of transfected mammalian cells for high content screening microscopy.
Methods in enzymology, 2005Co-Authors: Holger Erfle, Rainer PepperkokAbstract:In this chapter we describe protocols for Reverse Transfection to generate mammalian cell arrays for systematic gene knock-downs by RNAi or knock-ins by ectopic cDNA expression. The method is suitable for high content screening microscopy at a high spatial and temporal resolution allowing even time-lapse analysis of hundreds of samples in parallel.
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siRNA cell arrays for high-content screening microscopy
BioTechniques, 2004Co-Authors: Holger Erfle, Jeremy C. Simpson, Philippe I. H. Bastiaens, Rainer PepperkokAbstract:RNA interference (RNAi) is a recent advance that provides the possibility to reduce the expression of specific target genes in cultured mammalian cells with potential applications on a genome-wide scale. However, to achieve this, robust methodologies that allow automated and efficient delivery of small interfering RNAs (siRNAs) into living cultured cells and reliable quality control of siRNA function must be in place. Here we describe the production of cell arrays for Reverse Transfection of tissue culture cells with siRNA and plasmid DNA suitable for subsequent high-content screening microscopy applications. All the necessary Transfection components are mixed prior to the robotic spotting on noncoated chambered coverglass tissue culture dishes, which are ideally suited for time-lapse microscopy applications in living cells. The addition of fibronectin to the spotting solution improves cell adherence. After cell seeding, no further cell culture manipulations, such as medium changes or the addition of 7 serum, are needed. Adaptation of the cell density improves autofocus performance for high-quality data acquisition and cell recognition. The co-Transfection of a nonspecific fluorescently labeled DNA oligomer with the specific siRNA helps to mark each successfully transfected cell and cell cluster. We demonstrate such an siRNA cell array in a microscope-based functional assay in living cells to determine the effect of various siRNA oligonucleotides against endogenous targets on cellular secretion.
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ISBI - Time-lapse microscopy-based genome wide RNAi screening in live human cells
3rd IEEE International Symposium on Biomedical Imaging: Macro to Nano 2006., 1Co-Authors: Beate Neumann, Holger Erfle, Phill Rogers, Rainer Pepperkok, Urban Liebel, Michael Held, Jan EllenbergAbstract:Specific knock-down of gene expression by RNA interference is the method of choice to study gene function in human cells. A uniquely detailed phenotypic readout of such hypomorphs is possible by live cell microscopy of appropriate fluorescent reporter proteins. Here, I will present a fully automated method for RNAi screens in cultured human cells, combining Reverse Transfection by siRNA cell arrays, automated time-lapse fluorescence microscopy and computational phenotype analysis by image processing. The strategy is illustrated using an automatically scored mitosis assay and provides an easily scalable platform that we currently use in genome-wide RNAi screens for several cellular functions.
Holger Erfle - One of the best experts on this subject based on the ideXlab platform.
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solid phase Reverse Transfection for intracellular delivery of functionally active proteins
Genome Research, 2017Co-Authors: Ruben Bulkescher, Vytaute Starkuviene, Holger ErfleAbstract:Delivery of large and functionally active biomolecules across cell membranes presents a challenge in cell biological experimentation. For this purpose, we developed a novel solid-phase Reverse Transfection method that is suitable for the intracellular delivery of proteins into mammalian cells with preservation of their function. We show results for diverse application areas of the method, ranging from antibody-mediated inhibition of protein function to CRISPR/Cas9-based gene editing in living cells. Our method enables prefabrication of "ready to transfect" substrates carrying diverse proteins. This allows their easy distribution and standardization of biological assays across different laboratories.
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Next-generation 9216-microwell cell arrays for high-content screening microscopy
BioTechniques, 2009Co-Authors: Jürgen Reymann, Nina Beil, Jürgen Beneke, Peter-paul Kaletta, Klaus Burkert, Holger ErfleAbstract:Reverse Transfection on cell arrays is a high-throughput method for the parallel Transfection of mammalian cells for use in high-content screening light microscopy. Here, we present novel 9216-microwell cell arrays which combine the advantages of multiwell plates (physically separated samples) and cell microarrays (high sample density and long-term storage).
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Work flow for multiplexing siRNA assays by solid-phase Reverse Transfection in multiwell plates.
Journal of biomolecular screening, 2008Co-Authors: Holger Erfle, Beate Neumann, Phill Rogers, Jutta Bulkescher, Jan Ellenberg, Rainer PepperkokAbstract:Solid-phase Reverse Transfection on cell microarrays is a high-throughput method for the parallel Transfection of mammalian cells. However, the cells transfected in this way have been restricted so far to microscopy-based analyses. Analysis methods such as Reverse transcriptase–polymerase chain reaction (RT-PCR) and access to higher cell numbers for statistical reasons in microscopy-based assays are not possible with solid-phase Reverse Transfection on cell microarrays. We have developed a quick and reliable protocol for automated solid-phase Reverse Transfection of human cells with siRNAs in multiwell plates complementing solid-phase Reverse Transfection on cell microarrays. The method retains all advantages of solid-phase Reverse Transfection such as long-term storage capacity after fabrication, reduced cytotoxicity, and reduced cost per screen compared with liquid-phase Transfection in multiwell plates. The protocol has been tested for the RNAi-mediated knockdown of several genes in different cell lines including U20S, RPE1, A549, and HeLa cells. We show that even 3 months after production of the “ready to transfect” multiwell plates, there is no reduction in their Transfection efficiency as assessed by RT-PCR and nuclear phenotyping by fluorescence microscopy. We conclude that solidphase Reverse Transfection in multiwell plates is a cost-efficient and flexible tool for multiplexing cellular assays. (Journal of Biomolecular Screening. 2008:575-580)
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Systematische Untersuchung von Kombinationswirkungen der HHV-8-Gene auf die NF-κB-Aktivierung
Chirurgisches Forum 2008, 2008Co-Authors: Andreas Konrad, Holger Erfle, Effi Wies, Mathias Thurau, Gaby Sander, Rene Leubert, Elisabeth Naschberger, B. Brors, R. Eils, Frank NeipelAbstract:Activation of nuclear factor-kappa B (NF-κB), a key regulatory molecule in inflammation, is essential for survival of Human Herpesvirus (HHV)-8-infected cells. Using Reverse Transfection cell arrays (RTCA) as an unbiased systems biology approach the effects of all HHV-8-encoded genes individually and of pairwise combinations of all K- and latent genes on NF-κB-activation were investigated.
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Reverse Transfection on cell arrays for high content screening microscopy.
Nature protocols, 2007Co-Authors: Holger Erfle, Beate Neumann, Phill Rogers, Jan Ellenberg, Urban Liebel, Michael Held, Thomas Walter, Rainer PepperkokAbstract:Here, we describe a robust protocol for the Reverse Transfection of cells on small interfering (siRNA) arrays, which, in combination with multi-channel immunofluorescence or time-lapse microscopy, is suitable for genome-wide RNA interference (RNAi) screens in intact human cells. The automatic production of 48 'Transfection ready' siRNA arrays, each containing 384 samples, takes in total 7 h. Pre-fabricated siRNA arrays can be used without loss of Transfection efficiency at least up to 15 months after printing. Different human cell lines that have been successfully transfected using the protocol are presented here. The present protocol has been applied to two genome-wide siRNA screens addressing mitosis and constitutive protein secretion.
Erica Ueda - One of the best experts on this subject based on the ideXlab platform.
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Superhydrophilic–Superhydrophobic Patterned Surfaces as High-Density Cell Microarrays: Optimization of Reverse Transfection
Advanced healthcare materials, 2016Co-Authors: Erica Ueda, Wenqian Feng, Pavel A. LevkinAbstract:High-density microarrays can screen thousands of genetic and chemical probes at once in a miniaturized and parallelized manner, and thus are a cost-effective alternative to microwell plates. Here, high-density cell microarrays are fabricated by creating superhydrophilic-superhydrophobic micropatterns in thin, nanoporous polymer substrates such that the superhydrophobic barriers confine both aqueous solutions and adherent cells within each superhydrophilic microspot. The superhydrophobic barriers confine and prevent the mixing of larger droplet volumes, and also control the spreading of droplets independent of the volume, minimizing the variability that arises due to different liquid and surface properties. Using a novel liposomal Transfection reagent, ScreenFect A, the method of Reverse cell Transfection is optimized on the patterned substrates and several factors that affect Transfection efficiency and cytotoxicity are identified. Higher levels of Transfection are achieved on HOOC- versus NH2 -functionalized superhydrophilic spots, as well as when gelatin and fibronectin are added to the Transfection mixture, while minimizing the amount of Transfection reagent improves cell viability. Almost no diffusion of the printed Transfection mixtures to the neighboring microspots is detected. Thus, superhydrophilic-superhydrophobic patterned surfaces can be used as cell microarrays and for optimizing Reverse cell Transfection conditions before performing further cell screenings.
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superhydrophilic superhydrophobic patterned surfaces as high density cell microarrays optimization of Reverse Transfection
Advanced Healthcare Materials, 2016Co-Authors: Erica Ueda, Wenqian Feng, Pavel A. LevkinAbstract:High-density microarrays can screen thousands of genetic and chemical probes at once in a miniaturized and parallelized manner, and thus are a cost-effective alternative to microwell plates. Here, high-density cell microarrays are fabricated by creating superhydrophilic-superhydrophobic micropatterns in thin, nanoporous polymer substrates such that the superhydrophobic barriers confine both aqueous solutions and adherent cells within each superhydrophilic microspot. The superhydrophobic barriers confine and prevent the mixing of larger droplet volumes, and also control the spreading of droplets independent of the volume, minimizing the variability that arises due to different liquid and surface properties. Using a novel liposomal Transfection reagent, ScreenFect A, the method of Reverse cell Transfection is optimized on the patterned substrates and several factors that affect Transfection efficiency and cytotoxicity are identified. Higher levels of Transfection are achieved on HOOC- versus NH2 -functionalized superhydrophilic spots, as well as when gelatin and fibronectin are added to the Transfection mixture, while minimizing the amount of Transfection reagent improves cell viability. Almost no diffusion of the printed Transfection mixtures to the neighboring microspots is detected. Thus, superhydrophilic-superhydrophobic patterned surfaces can be used as cell microarrays and for optimizing Reverse cell Transfection conditions before performing further cell screenings.