scholarly journals Automated High-Throughput Flow Cytometry for High-Content Screening in Antibody Development

2018 ◽  
Vol 23 (7) ◽  
pp. 656-666 ◽  
Author(s):  
Yana Wang ◽  
Tomoki Yoshihara ◽  
Samson King ◽  
Tinh Le ◽  
Patrick Leroy ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Sample Preparation ◽  
High Throughput ◽  
Screening Method ◽  
Therapeutic Modality ◽  
Antibody Concentration ◽  
Lead Discovery ◽  
Multiple Parameters ◽  
Antibody Discovery ◽  
Multiple Cell

The tedious sample preparation for flow cytometry limits the throughput and thus its usage as a primary screening method despite its sensitivity and accuracy. With the growing focus on utilizing antibodies as a therapeutic modality in drug discovery, it is critical to develop a high-throughput flow cytometry (HTFC) workflow to cope with the increasing need to support antibody discovery programs. We have developed a seamless HTFC sample preparation and readout workflow using the HighRes modular robotic system and the IntelliCyt iQue Screener PLUS. To fully utilize the advantages offered by flow cytometry, we typically multiplex multiple cell lines of interest in one well to simultaneously quantitate on-target activity and nonspecific activity along with measurement of antibody concentration. The ability to measure multiple parameters coupled with speed and increased accuracy provides gains in productivity and helps speed up antibody lead discovery.

scholarly journals High-throughput ultrastructure screening using electron microscopy and fluorescent barcoding

2019 ◽  
Vol 218 (8) ◽  
pp. 2797-2811 ◽  
Author(s):  
Yury S. Bykov ◽  
Nir Cohen ◽  
Natalia Gabrielli ◽  
Hetty Manenschijn ◽  
Sonja Welsch ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Sample Preparation ◽  
High Throughput ◽  
Fluorescent Microscopy ◽  
Large Datasets ◽  
Genetic Screens ◽  
Cell Type ◽  
Resolution Limit ◽  
Computer Image Analysis ◽  
Multiple Cell

Genetic screens using high-throughput fluorescent microscopes have generated large datasets, contributing many cell biological insights. Such approaches cannot tackle questions requiring knowledge of ultrastructure below the resolution limit of fluorescent microscopy. Electron microscopy (EM) reveals detailed cellular ultrastructure but requires time-consuming sample preparation, limiting throughput. Here we describe a robust method for screening by high-throughput EM. Our approach uses combinations of fluorophores as barcodes to uniquely mark each cell type in mixed populations and correlative light and EM (CLEM) to read the barcode of each cell before it is imaged by EM. Coupled with an easy-to-use software workflow for correlation, segmentation, and computer image analysis, our method, called “MultiCLEM,” allows us to extract and analyze multiple cell populations from each EM sample preparation. We demonstrate several uses for MultiCLEM with 15 different yeast variants. The methodology is not restricted to yeast, can be scaled to higher throughput, and can be used in multiple ways to enable EM to become a powerful screening technique.

A simple, highly sensitive, high throughput and organic solvent-free screening method for melamine by microsphere-based flow cytometry immunoassay

2015 ◽  
Vol 7 (14) ◽  
pp. 5989-5995 ◽  
Author(s):  
Tik-Hung Tsoi ◽  
Wing-Tak Wong
Keyword(s):  
Flow Cytometry ◽  
Organic Solvent ◽  
High Throughput ◽  
Screening Method ◽  
Solvent Free ◽  
Highly Sensitive

A facile method based on indirect competitive inhibitory microsphere-based flow cytometry for melamine screening.

Abstract 5774: Novel cell-based high-throughput hybridoma screening method using the Celigo image cytometer for antibody discovery

2018 ◽  
Author(s):  
Leo L. Chan ◽  
Haohai Zhang ◽  
William Rice ◽  
Nasim Kassam ◽  
Maria S. Longhi ◽  
...  
Keyword(s):  
High Throughput ◽  
Screening Method ◽  
Antibody Discovery

Su.83. High Throughput Sample Preparation in 96 Well Plates for Flow Cytometry Analysis

2008 ◽  
Vol 127 ◽  
pp. S151
Author(s):  
Carlos Aparicio ◽  
Julie Wilkinson ◽  
Ileana Munoz-Antoni ◽  
Enrique Rabellino
Keyword(s):  
Flow Cytometry ◽  
Sample Preparation ◽  
High Throughput ◽  
Flow Cytometry Analysis

scholarly journals A Scalable Pipeline for High-Throughput Flow Cytometry

2018 ◽  
Vol 23 (7) ◽  
pp. 708-718
Author(s):  
Aaron C. Wilson ◽  
Ioannis K. Moutsatsos ◽  
Gary Yu ◽  
Javier J. Pineda ◽  
Yan Feng ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Sample Preparation ◽  
High Throughput ◽  
Test Sample ◽  
Molecular Probe ◽  
Phenotypic Analysis ◽  
Mhc I ◽  
Level Data ◽  
Well Assay

Flow cytometry (FC) provides high-content data for a variety of applications, including phenotypic analysis of cell surface and intracellular markers, characterization of cell supernatant or lysates, and gene expression analysis. Historically, sample preparation, acquisition, and analysis have presented as a bottleneck for running such types of assays at scale. This article will outline the solutions that have been implemented at Novartis which have allowed high-throughput FC to be successfully conducted and analyzed for a variety of cell-based assays. While these experiments were generally conducted to measure phenotypic responses from a well-characterized and information-rich small molecular probe library known as the Mechanism-of-Action (MoA) Box, they are broadly applicable to any type of test sample. The article focuses on application of automated methods for FC sample preparation in 384-well assay plates. It also highlights a pipeline for analyzing large volumes of FC data, covering a visualization approach that facilitates review of screen-level data by dynamically embedding FlowJo (FJ) workspace images for each sample into a Spotfire file, directly linking them to the metric being observed. Finally, an application of these methods to a screen for MHC-I expression upregulators is discussed.

Directed Evolution of P450 Fatty Acid Decarboxylases via High-Throughput Screening Towards Improved Catalytic Activity

2019 ◽  
Author(s):  
Huifang Xu ◽  
Weinan Liang ◽  
Linlin Ning ◽  
Yuanyuan Jiang ◽  
Wenxia Yang ◽  
...  
Keyword(s):  
Catalytic Activity ◽  
Fatty Acid ◽  
Directed Evolution ◽  
High Throughput ◽  
High Throughput Screening ◽  
Colorimetric Detection ◽  
Screening Method ◽  
Random Mutagenesis ◽  
Direct Production ◽  
Consumption Activity

P450 fatty acid decarboxylases (FADCs) have recently been attracting considerable attention owing to their one-step direct production of industrially important 1-alkenes from biologically abundant feedstock free fatty acids under mild conditions. However, attempts to improve the catalytic activity of FADCs have met with little success. Protein engineering has been limited to selected residues and small mutant libraries due to lack of an effective high-throughput screening (HTS) method. Here, we devise a catalase-deficient <i>Escherichia coli</i> host strain and report an HTS approach based on colorimetric detection of H<sub>2</sub>O<sub>2</sub>-consumption activity of FADCs. Directed evolution enabled by this method has led to effective identification for the first time of improved FADC variants for medium-chain 1-alkene production from both DNA shuffling and random mutagenesis libraries. Advantageously, this screening method can be extended to other enzymes that stoichiometrically utilize H<sub>2</sub>O<sub>2</sub> as co-substrate.

High-Throughput, Site-Specific Sample Prep of Ultra-Thin TEM Lamella for Process Metrology and Failure Analysis

2012 ◽  
Author(s):  
Roger Alvis ◽  
Jeff Blackwood ◽  
Sang-Hoon Lee ◽  
Matthew Bray
Keyword(s):  
Sample Preparation ◽  
Failure Analysis ◽  
Process Monitoring ◽  
High Throughput ◽  
Focused Ion Beam ◽  
Ion Beam ◽  
Memory Devices ◽  
Site Specific ◽  
Critical Dimensions ◽  
Tem Lamella

Abstract Semiconductor devices with critical dimensions less than 20nm are now being manufactured in volume. A challenge facing the failure analysis and process-monitoring community is two-fold. The first challenge of TEM sample prep of such small devices is that the basic need to end-point on a feature-of-interest pushes the imaging limit of the instrument being used to prepare the lamella. The second challenge posed by advanced devices is to prepare an artifact-free lamella from non-planar devices such as finFETs as well as from structures incorporating ‘non-traditional’ materials. These challenges are presently overcome in many advanced logic and memory devices in the focused ion beam-based TEM sample preparation processes by inverting the specimen prior to thinning to electron transparency. This paper reports a highthroughput method for the routine preparation of artifact-free TEM lamella of 20nm thickness, or less.

scholarly journals Development of a Novel High-Throughput Screen and Identification of Small-Molecule Inhibitors of the Gα–RGS17 Protein–Protein Interaction Using AlphaScreen

2011 ◽  
Vol 16 (8) ◽  
pp. 869-877 ◽  
Author(s):  
Duncan I. Mackie ◽  
David L. Roman
Keyword(s):  
Small Molecule ◽  
High Throughput ◽  
Protein Interaction ◽  
High Throughput Screening ◽  
Drug Targets ◽  
Screening Method ◽  
Fold Increase ◽  
Rgs Proteins ◽  
High Throughput Screen ◽  
Protein Protein Interaction

In this study, the authors used AlphaScreen technology to develop a high-throughput screening method for interrogating small-molecule libraries for inhibitors of the Gαo–RGS17 interaction. RGS17 is implicated in the growth, proliferation, metastasis, and the migration of prostate and lung cancers. RGS17 is upregulated in lung and prostate tumors up to a 13-fold increase over patient-matched normal tissues. Studies show RGS17 knockdown inhibits colony formation and decreases tumorigenesis in nude mice. The screen in this study uses a measurement of the Gαo–RGS17 protein–protein interaction, with an excellent Z score exceeding 0.73, a signal-to-noise ratio >70, and a screening time of 1100 compounds per hour. The authors screened the NCI Diversity Set II and determined 35 initial hits, of which 16 were confirmed after screening against controls. The 16 compounds exhibited IC50 <10 µM in dose–response experiments. Four exhibited IC50 values <6 µM while inhibiting the Gαo–RGS17 interaction >50% when compared to a biotinylated glutathione-S-transferase control. This report describes the first high-throughput screen for RGS17 inhibitors, as well as a novel paradigm adaptable to many other RGS proteins, which are emerging as attractive drug targets for modulating G-protein-coupled receptor signaling.

scholarly journals A high-throughput screening method for evolving a demethylase enzyme with improved and new functionalities

2020 ◽  
Author(s):  
Yuru Wang ◽  
Christopher D Katanski ◽  
Christopher Watkins ◽  
Jessica N Pan ◽  
Qing Dai ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Screening Method ◽  
Targeted Mutagenesis ◽  
Rna Repair ◽  
Dna And Rna ◽  
Modified Dna ◽  
Dna Substrates ◽  
Trna Sequencing

Abstract AlkB is a DNA/RNA repair enzyme that removes base alkylations such as N1-methyladenosine (m1A) or N3-methylcytosine (m3C) from DNA and RNA. The AlkB enzyme has been used as a critical tool to facilitate tRNA sequencing and identification of mRNA modifications. As a tool, AlkB mutants with better reactivity and new functionalities are highly desired; however, previous identification of such AlkB mutants was based on the classical approach of targeted mutagenesis. Here, we introduce a high-throughput screening method to evaluate libraries of AlkB variants for demethylation activity on RNA and DNA substrates. This method is based on a fluorogenic RNA aptamer with an internal modified RNA/DNA residue which can block reverse transcription or introduce mutations leading to loss of fluorescence inherent in the cDNA product. Demethylation by an AlkB variant eliminates the blockage or mutation thereby restores the fluorescence signals. We applied our screening method to sites D135 and R210 in the Escherichia coli AlkB protein and identified a variant with improved activity beyond a previously known hyperactive mutant toward N1-methylguanosine (m1G) in RNA. We also applied our method to O6-methylguanosine (O6mG) modified DNA substrates and identified candidate AlkB variants with demethylating activity. Our study provides a high-throughput screening method for in vitro evolution of any demethylase enzyme.

Sign in / Sign up

Export Citation Format

Share Document