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

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…
Yana Wang, Tomoki Yoshihara, Samson King, Ching Kit Chan

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Comparing Flow Cytometry QBeads PlexScreen Assays with Other Immunoassays for Determining Multiple Analytes

Immunoassays, utilizing the affinity of antibodies to their antigens, are powerful techniques and have been widely used for quantifying analytes, such as cytokines, in biological samples in the clinic and in drug discovery. Various immunoassays have been developed to fit for different purposes. Recently, bead-based flow cytometry assays have emerged…
Mei Ding, Anders Cavallin, Nils-Olov Hermansson

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High-Throughput Flow Cytometric Method for the Simultaneous Measurement of CAR-T Cell Characterization and Cytotoxicity against Solid Tumor Cell Lines

High-throughput flow cytometry is an attractive platform for the analysis of adoptive cellular therapies such as chimeric antigen receptor T cell therapy (CAR-T) because it allows for the concurrent measurement of T cell-dependent cellular cytotoxicity (TDCC) and the functional characterization of engineered T cells with respect to percentage of CAR…
Emily M Martinez, Samuel D Klebanoff, Stephanie Secrest

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A Multiplexed Screening Assay to Evaluate Chemotherapy-Induced Myelosuppression Using Healthy Peripheral Blood and Bone Marrow

Myelosuppression is a major side effect of chemotherapy in cancer patients and can result in infections, bleeding complications, and increased risk of morbidity and mortality, as well as limit the drug dose and frequency of administration. Chemotherapy-induced myelosuppression is caused by the disruption of normal hematopoiesis. Thus, prior understanding of…
Komal K Javarappa, Dimitrios Tsallos

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Reduction of Nonspecificity Motifs in Synthetic Antibody Libraries

Successful antibody development requires both functional binding and desirable biophysical characteristics. In the current study, we analyze the causes of one hurdle to clinical development, off-target reactivity, or nonspecificity. We used a high-throughput nonspecificity assay to isolate panels of nonspecific antibodies from two synthetic single-chain variable fragment libraries expressed on…
Ryan L Kelly, Doris Le, Jessie Zhao

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Regulated Expansion and Survival of Chimeric Antigen Receptor-Modified T Cells Using Small Molecule-Dependent Inducible MyD88/CD40

Anti-tumor efficacy of T cells engineered to express chimeric antigen receptors (CARs) is dependent on their specificity, survival, and in vivo expansion following adoptive transfer. Toll-like receptor (TLR) and CD40 signaling in T cells can improve persistence and drive proliferation of antigen-specific CD4+ and CD8+ T cells following pathogen challenge…
Aaron E Foster, Aruna Mahendravada, Nicholas P Shinners

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Cytosolic delivery of siRNA by ultra-high affinity dsRNA binding proteins

Protein-based methods of siRNA delivery are capable of uniquely specific targeting, but are limited by technical challenges such as low potency or poor biophysical properties. Here, we engineered a series of ultra-high affinity siRNA binders based on the viral protein p19 and developed them into siRNA carriers targeted to the…
Nicole J. Yang, Monique J. Kauke, Fangdi Sun

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Dynamic cybergenetic control of bacterial co-culture composition via optogenetic feedback

Communities of microbes play important roles in natural environments and hold great potential for deploying division-of-labor strategies in synthetic biology and bioproduction. However, the difficulty of controlling the composition of microbial consortia over time hinders their optimal use in many applications. Here, we present a fully automated, high-throughput platform that…
Joaquín Gutiérrez Mena, Sant Kumar

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Radioiodination of extravesicular surface constituents to study the biocorona, cell trafficking and storage stability of extracellular vesicles

Extracellular vesicles (EVs) are produced by all cell types and serve as biological packets delivering a wide variety of molecules for cell-to-cell communication. However, the biology of the EV extravesicular surface domain that we have termed EV ‘biocorona’ remains underexplored. Upon cell secretion, EVs possess an innate biocorona containing membrane…
Saigopalakrishna S. Yerneni

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Mutational sources of trans-regulatory variation affecting gene expression in Saccharomyces cerevisiae

Heritable variation in a gene’s expression arises from mutations impacting cis- and trans-acting components of its regulatory network. Here, we investigate how trans-regulatory mutations are distributed within the genome and within a gene regulatory network by identifying and characterizing 69 mutations with trans-regulatory effects on expression of the same focal…
Crisandra J Diaz, Elizabeth A Walker, Stephen Tryban

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