scholarly journals High-throughput screening and validation of antibodies against synaptic proteins to explore opioid signaling dynamics

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Mariana Lemos Duarte ◽  
Nikita A. Trimbake ◽  
Achla Gupta ◽  
Christine Tumanut ◽  
Xiaomin Fan ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Temporal Dynamics ◽  
Single Cells ◽  
Synaptic Proteins ◽  
Functional Screening ◽  
Signaling Dynamics ◽  
Antibody Libraries ◽  
Multiple State ◽  
Opioid Signaling

AbstractAntibodies represent powerful tools to examine signal transduction pathways. Here, we present a strategy integrating multiple state-of-the-art methods to produce, validate, and utilize antibodies. Focusing on understudied synaptic proteins, we generated 137 recombinant antibodies. We used yeast display antibody libraries from the B cells of immunized rabbits, followed by FACS sorting under stringent conditions to identify high affinity antibodies. The antibodies were validated by high-throughput functional screening, and genome editing. Next, we explored the temporal dynamics of signaling in single cells. A subset of antibodies targeting opioid receptors were used to examine the effect of treatment with opiates that have played central roles in the worsening of the ‘opioid epidemic.’ We show that morphine and fentanyl exhibit differential temporal dynamics of receptor phosphorylation. In summary, high-throughput approaches can lead to the identification of antibody-based tools required for an in-depth understanding of the temporal dynamics of opioid signaling.

scholarly journals Raman-Activated Droplet Sorting (RADS) for Label-Free High-Throughput Screening of Microalgal Single-Cells

2017 ◽  
Vol 89 (22) ◽  
pp. 12569-12577 ◽  
Author(s):  
Xixian Wang ◽  
Lihui Ren ◽  
Yetian Su ◽  
Yuetong Ji ◽  
Yaoping Liu ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Single Cells ◽  
Label Free ◽  
Droplet Sorting

scholarly journals Assay Establishment and Validation of a High-Throughput Screening Platform for Three-Dimensional Patient-Derived Colon Cancer Organoid Cultures

2016 ◽  
Vol 21 (9) ◽  
pp. 931-941 ◽  
Author(s):  
Karsten Boehnke ◽  
Philip W. Iversen ◽  
Dirk Schumacher ◽  
María José Lallena ◽  
Rubén Haro ◽  
...  
Keyword(s):  
Colon Cancer ◽  
Drug Discovery ◽  
High Throughput ◽  
High Throughput Screening ◽  
Single Cells ◽  
Three Dimensional ◽  
Compound Screening ◽  
Organoid Cultures ◽  
Screening Platform ◽  
Disease Specific

The application of patient-derived three-dimensional culture systems as disease-specific drug sensitivity models has enormous potential to connect compound screening and clinical trials. However, the implementation of complex cell-based assay systems in drug discovery requires reliable and robust screening platforms. Here we describe the establishment of an automated platform in 384-well format for three-dimensional organoid cultures derived from colon cancer patients. Single cells were embedded in an extracellular matrix by an automated workflow and subsequently self-organized into organoid structures within 4 days of culture before being exposed to compound treatment. We performed validation of assay robustness and reproducibility via plate uniformity and replicate-experiment studies. After assay optimization, the patient-derived organoid platform passed all relevant validation criteria. In addition, we introduced a streamlined plate uniformity study to evaluate patient-derived colon cancer samples from different donors. Our results demonstrate the feasibility of using patient-derived tumor samples for high-throughput assays and their integration as disease-specific models in drug discovery.

scholarly journals A high throughput bispecific antibody discovery pipeline

2021 ◽  
Author(s):  
Aude I. Segaliny ◽  
Jayapriya Jayaraman ◽  
Xiaoming Chen ◽  
Jonathan Chong ◽  
Ryan Luxon ◽  
...  
Keyword(s):  
Single Cell ◽  
High Throughput ◽  
High Throughput Screening ◽  
In Vitro Cytotoxicity ◽  
Functional Screening ◽  
Sequence Composition ◽  
Single Cell Pcr ◽  
Screening Efficiency ◽  
And Function

AbstractBispecific antibodies (BsAbs) represent an emerging class of immunotherapy but inefficiency in the current BsAb discovery paradigm has limited their broad clinical availability. Here we report a high throughput, agnostic, single-cell-based BsAb functional screening pipeline, comprising molecular and cell engineering for efficient generation of BsAb library cells, followed by functional interrogation at the single-cell level to identify and sort positive clones and downstream sequence identification with single-cell PCR and sequencing and functionality characterization. Using a CD19xCD3 bispecific T cell engager (BiTE) as a model system, we demonstrate that our single cell platform possesses a high throughput screening efficiency of up to one and half million variant library cells per run and can isolate rare functional clones at low abundance of 0.008%. Using a complex CD19xCD3 BiTE-expressing cell library with approximately 22,300 unique variants comprising combinatorially varied scFvs, connecting linkers and VL/VH orientations, we have identified 98 unique clones including extremely rare ones (∼ 0.001% abundance). We also discovered BiTEs that exhibit novel properties contradictory to conventional wisdom, including harboring rigid scFv connecting peptide linkers yet with in vitro cytotoxicity comparable to that of clinically approved Blinatumomab. Through sequencing analyses on sorted BiTE clones, we discovered multiple design variable preferences for functionality including the CD19VL-VH– CD3VH-VL and CD19VH-VL–CD3VH-VL arrangements being the most favored orientation. Sequence analysis further interrogated the sequence composition of the CDRH3 domain in scFvs and identified amino acid residues conserved for function. We expect our single cell platform to not only significantly increase the development speed of high quality of new BsAb therapeutics for cancer and other disorders, but also enable identifying generalizable design principles for new BsAbs and other immunotherapeutics based on an in-depth understanding of the inter-relationships between sequence, structure, and function.

Abstract 15707: Fluorescence Voltage Recordings From Stem Cell-Derived Cardiomyocytes Enable High-Throughput Screening for Cardiotoxicity

Circulation ◽  
2014 ◽  
Vol 130 (suppl_2) ◽  
Author(s):  
Jordan S Leyton-Mange ◽  
Robert W Mills ◽  
Min-Young Jang ◽  
Xaio Ling ◽  
Patrick T Ellinor ◽  
...  
Keyword(s):  
Stem Cell ◽  
High Throughput ◽  
High Throughput Screening ◽  
High Speed ◽  
Single Cells ◽  
Drug Application ◽  
Negative Control ◽  
Optical Measurements ◽  
Drug Induced ◽  
Dose Dependent

Introduction: The lack of high quality predictive models for drug-induced QT prolongation continues to be a significant problem in pharmaceutical development. While human pluripotent stem cell derived-cardiomyocytes (hPSC-CMs) hold promise to be a valuable tool for drug discovery, efforts have been frustrated by the labor-intensive nature of electrophysiological recordings and the heterogeneity of hPSC-CMs populations. Methods: Using lentivirus, we introduced the genetically encoded fluorescent voltage reporter, A242-Arclight, into hPSC-CM monolayers in multi-well plates. An inverted fluorescence microscope was fit with an environmentally controlled enclosure and automated stage. High speed imaging with a Photometrics Evolve 128 EMCCD camera was performed at baseline and after administration of test compounds. Optical traces were processed using a custom program and composite AP durations, APD80, were compared before and after drug application (Figures A & B). Results: Baseline APD80 values displayed high degree of consistency between wells: 483±59 msec. High-throughput data acquisition demonstrated dose dependent APD80 increases from all QT-prolonging agents tested as well as dose dependent APD80 decrease from pinacidil. In contrast, negative control compounds caused no significant changes in APD80. Results from a representative plate are shown (Figure C). Conclusions: Optical measurements provide rapid recordings of drug-induced AP duration changes, and offer a strategy to non-invasively screen hPSC-CMs in high-throughput. Recording from cell monolayers as opposed to single cells and using paired comparisons may be beneficial in addressing the heterogeneity amongst hPSC-CM preparations.

scholarly journals Integration of Droplet Microfluidic Tools for Single-cell Functional Metagenomics: An Engineering Head Start

2021 ◽  
Author(s):  
David Conchouso ◽  
Amani Al-Ma’abadi ◽  
Hayedeh Behzad ◽  
Mohammed Alarawi ◽  
Masahito Hosokawa ◽  
...  
Keyword(s):  
Head Start ◽  
Single Cell ◽  
High Throughput ◽  
Single Cells ◽  
Droplet Microfluidics ◽  
Metagenomic Data ◽  
Functional Screening ◽  
Functional Metagenomics ◽  
Technical Details ◽  
Droplet Sorting

<p>Droplet microfluidics techniques have shown promising results to study single-cells at high throughput. However, their adoption in laboratories studying “-omics” sciences is still irrelevant because of the field’s complex and multidisciplinary nature. To facilitate their use, here we provide engineering details and organized protocols for integrating three droplet-based microfluidic technologies into the metagenomic pipeline to enable functional screening of bioproducts at high throughput. First, a device encapsulating single-cells in droplets at a rate of ~ 250 Hz is described considering droplet size and cell growth. Then, we expand on previously reported fluorescent activated droplet sorting (FADS) systems to integrate the use of 4 independent fluorescence-exciting lasers (e.g., 405, 488, 561, 637 nm) in a single platform to make it compatible with different fluorescence-emitting biosensors. For this sorter, both hardware and software are provided and optimized for effortlessly sorting droplets at 60 Hz. Then, a passive droplet merger was also integrated into our method to enable adding new reagents to already made droplets at a rate of 200 Hz. Finally, we provide an optimized recipe for manufacturing these chips using silicon dry-etching tools. Because of the overall integration and the technical details presented here, our approach allows biologists to quickly use microfluidic technologies and achieve both single-cell resolution and high-throughput (> 50,000 cells/day) capabilities to mining and bioprospecting metagenomic data.</p>

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