P2-201: FLOW CYTOMETRY DETECTION OF MULTIPLE CELL TYPES AND EXOSOMAL ISOLATION FROM AD CORTEX

Cell quantification assays are essential components of most biological and clinical labs. However, many currently available quantification assays, including flow cytometry and commercial cell counting systems, suffer from unique drawbacks that limit their overall efficacy. In order to address the shortcomings of traditional quantification assays, we have designed a robust, low-cost, automated optical cell cytometer that quantifies individual cells in a multiwell plate using tools readily available in most labs. Plating and subsequent quantification of various dilution series using the automated optical cytometer demonstrates the single-cell sensitivity, near-perfect R2 accuracy, and greater than 5-log dynamic range of our system. Further, the optical cytometer is capable of obtaining absolute counts of multiple cell types in one well as part of a co-culture setup. To demonstrate this ability, we recreated an experiment that assesses the tumoricidal properties of primed macrophages on co-cultured tumor cells as a proof-of-principle test. The results of the experiment reveal that primed macrophages display enhanced cytotoxicity towards tumor cells while simultaneously losing the ability to proliferate, an example of a dynamic interplay between two cell populations that our optical cytometer is successfully able to elucidate.

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A customizable microscopy system for the automated quantification and characterization of multiple adherent cell types: an alternative to flow cytometry

10.7287/peerj.preprints.26584v1 ◽

2018 ◽

Author(s):

Vishwaratn Asthana ◽

Yuqi Tang ◽

Adam Ferguson ◽

Pallavi Bugga ◽

Anantratn Asthana ◽

...

Keyword(s):

Flow Cytometry ◽

Tumor Cells ◽

Dynamic Range ◽

Low Cost ◽

Cell Types ◽

Cell Counting ◽

Adherent Cell ◽

Optical Cell ◽

Essential Components ◽

Multiple Cell

Cell quantification assays are essential components of most biological and clinical labs. However, many currently available quantification assays, including flow cytometry and commercial cell counting systems, suffer from unique drawbacks that limit their overall efficacy. In order to address the shortcomings of traditional quantification assays, we have designed a robust, low-cost, automated optical cell cytometer that quantifies individual cells in a multiwell plate using tools readily available in most labs. Plating and subsequent quantification of various dilution series using the automated optical cytometer demonstrates the single-cell sensitivity, near-perfect R2 accuracy, and greater than 5-log dynamic range of our system. Further, the optical cytometer is capable of obtaining absolute counts of multiple cell types in one well as part of a co-culture setup. To demonstrate this ability, we recreated an experiment that assesses the tumoricidal properties of primed macrophages on co-cultured tumor cells as a proof-of-principle test. The results of the experiment reveal that primed macrophages display enhanced cytotoxicity towards tumor cells while simultaneously losing the ability to proliferate, an example of a dynamic interplay between two cell populations that our optical cytometer is successfully able to elucidate.

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Faculty Opinions recommendation of IL-10 is up-regulated in multiple cell types during viremic HIV infection and reversibly inhibits virus-specific T cells.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.6294957.6383055 ◽

2010 ◽

Author(s):

Susana Asin ◽

Christiane Rollenhagen

Keyword(s):

T Cells ◽

Hiv Infection ◽

Cell Types ◽

Multiple Cell

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Multiple cell types contribute to the atherosclerotic lesion fibrous cap by PDGFRβ and bioenergetic mechanisms

Nature Metabolism ◽

10.1038/s42255-020-00338-8 ◽

2021 ◽

Vol 3 (2) ◽

pp. 166-181 ◽

Cited By ~ 2

Author(s):

Alexandra A. C. Newman ◽

Vlad Serbulea ◽

Richard A. Baylis ◽

Laura S. Shankman ◽

Xenia Bradley ◽

...

Keyword(s):

Atherosclerotic Lesion ◽

Cell Types ◽

Fibrous Cap ◽

Multiple Cell

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Mitoapocynin, a mitochondria targeted derivative of apocynin induces mitochondrial ROS generation and apoptosis in multiple cell types including cardiac myoblasts: a potential constraint to its therapeutic use

Molecular and Cellular Biochemistry ◽

10.1007/s11010-020-04039-4 ◽

2021 ◽

Author(s):

Amena Mahmood ◽

Padmini Bisoyi ◽

Rajkumar Banerjee ◽

Md Yousuf ◽

Shyamal K. Goswami

Keyword(s):

Cell Types ◽

Therapeutic Use ◽

Ros Generation ◽

Mitochondrial Ros ◽

Multiple Cell ◽

Cardiac Myoblasts

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MyD88 Is Not Required for Muscle Injury-Induced Endochondral Heterotopic Ossification in a Mouse Model of Fibrodysplasia Ossificans Progressiva

Biomedicines ◽

10.3390/biomedicines9060630 ◽

2021 ◽

Vol 9 (6) ◽

pp. 630

Author(s):

Huili Lyu ◽

Cody M. Elkins ◽

Jessica L. Pierce ◽

C. Henrique Serezani ◽

Daniel S. Perrien

Keyword(s):

Heterotopic Ossification ◽

Muscle Injury ◽

Cell Types ◽

Fibrodysplasia Ossificans Progressiva ◽

Inflammatory Signaling ◽

Conditional Deletion ◽

Pathway Crosstalk ◽

Multiple Cell

Excess inflammation and canonical BMP receptor (BMPR) signaling are coinciding hallmarks of the early stages of injury-induced endochondral heterotopic ossification (EHO), especially in the rare genetic disease fibrodysplasia ossificans progressiva (FOP). Multiple inflammatory signaling pathways can synergistically enhance BMP-induced Smad1/5/8 activity in multiple cell types, suggesting the importance of pathway crosstalk in EHO and FOP. Toll-like receptors (TLRs) and IL-1 receptors mediate many of the earliest injury-induced inflammatory signals largely via MyD88-dependent pathways. Thus, the hypothesis that MyD88-dependent signaling is required for EHO was tested in vitro and in vivo using global or Pdgfrα-conditional deletion of MyD88 in FOP mice. As expected, IL-1β or LPS synergistically increased Activin A (ActA)-induced phosphorylation of Smad 1/5 in fibroadipoprogenitors (FAPs) expressing Alk2R206H. However, conditional deletion of MyD88 in Pdgfrα-positive cells of FOP mice did not significantly alter the amount of muscle injury-induced EHO. Even more surprisingly, injury-induced EHO was not significantly affected by global deletion of MyD88. These studies demonstrate that MyD88-dependent signaling is dispensable for injury-induced EHO in FOP mice.

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Microtubule–microtubule sliding by kinesin-1 is essential for normal cytoplasmic streaming in Drosophila oocytes

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1522424113 ◽

2016 ◽

Vol 113 (34) ◽

pp. E4995-E5004 ◽

Cited By ~ 41

Author(s):

Wen Lu ◽

Michael Winding ◽

Margot Lakonishok ◽

Jill Wildonger ◽

Vladimir I. Gelfand

Keyword(s):

Cytoplasmic Streaming ◽

Cell Types ◽

Nurse Cells ◽

Wild Type ◽

Actin Cortex ◽

Microtubule Motor ◽

Multiple Cell ◽

Cytoplasmic Microtubules ◽

Two Populations

Cytoplasmic streaming in Drosophila oocytes is a microtubule-based bulk cytoplasmic movement. Streaming efficiently circulates and localizes mRNAs and proteins deposited by the nurse cells across the oocyte. This movement is driven by kinesin-1, a major microtubule motor. Recently, we have shown that kinesin-1 heavy chain (KHC) can transport one microtubule on another microtubule, thus driving microtubule–microtubule sliding in multiple cell types. To study the role of microtubule sliding in oocyte cytoplasmic streaming, we used a Khc mutant that is deficient in microtubule sliding but able to transport a majority of cargoes. We demonstrated that streaming is reduced by genomic replacement of wild-type Khc with this sliding-deficient mutant. Streaming can be fully rescued by wild-type KHC and partially rescued by a chimeric motor that cannot move organelles but is active in microtubule sliding. Consistent with these data, we identified two populations of microtubules in fast-streaming oocytes: a network of stable microtubules anchored to the actin cortex and free cytoplasmic microtubules that moved in the ooplasm. We further demonstrated that the reduced streaming in sliding-deficient oocytes resulted in posterior determination defects. Together, we propose that kinesin-1 slides free cytoplasmic microtubules against cortically immobilized microtubules, generating forces that contribute to cytoplasmic streaming and are essential for the refinement of posterior determinants.

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Single-cell mapping of focused ultrasound-transfected brain

Gene Therapy ◽

10.1038/s41434-021-00226-0 ◽

2021 ◽

Author(s):

A. S. Mathew ◽

C. M. Gorick ◽

R. J. Price

Keyword(s):

Single Cell ◽

Focused Ultrasound ◽

Cell Types ◽

Brain Cell ◽

Therapeutic Modality ◽

Full Potential ◽

Stress Genes ◽

Multiple Cell ◽

Differential Gene

AbstractGene delivery via focused ultrasound (FUS) mediated blood-brain barrier (BBB) opening is a disruptive therapeutic modality. Unlocking its full potential will require an understanding of how FUS parameters (e.g., peak-negative pressure (PNP)) affect transfected cell populations. Following plasmid (mRuby) delivery across the BBB with 1 MHz FUS, we used single-cell RNA-sequencing to ascertain that distributions of transfected cell types were highly dependent on PNP. Cells of the BBB (i.e., endothelial cells, pericytes, and astrocytes) were enriched at 0.2 MPa PNP, while transfection of cells distal to the BBB (i.e., neurons, oligodendrocytes, and microglia) was augmented at 0.4 MPa PNP. PNP-dependent differential gene expression was observed for multiple cell types. Cell stress genes were upregulated proportional to PNP, independent of cell type. Our results underscore how FUS may be tuned to bias transfection toward specific brain cell types in vivo and predict how those cells will respond to transfection.

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Outflow Tract Formation—Embryonic Origins of Conotruncal Congenital Heart Disease

Journal of Cardiovascular Development and Disease ◽

10.3390/jcdd8040042 ◽

2021 ◽

Vol 8 (4) ◽

pp. 42

Author(s):

Sonia Stefanovic ◽

Heather C. Etchevers ◽

Stéphane Zaffran

Keyword(s):

Congenital Heart ◽

Cardiac Development ◽

Heart Defects ◽

Dynamic Structure ◽

Cell Types ◽

Outflow Tract ◽

Heart Tube ◽

Heart Field ◽

Multiple Cell ◽

The Many

Anomalies in the cardiac outflow tract (OFT) are among the most frequent congenital heart defects (CHDs). During embryogenesis, the cardiac OFT is a dynamic structure at the arterial pole of the heart. Heart tube elongation occurs by addition of cells from pharyngeal, splanchnic mesoderm to both ends. These progenitor cells, termed the second heart field (SHF), were first identified twenty years ago as essential to the growth of the forming heart tube and major contributors to the OFT. Perturbation of SHF development results in common forms of CHDs, including anomalies of the great arteries. OFT development also depends on paracrine interactions between multiple cell types, including myocardial, endocardial and neural crest lineages. In this publication, dedicated to Professor Andriana Gittenberger-De Groot and her contributions to the field of cardiac development and CHDs, we review some of her pioneering studies of OFT development with particular interest in the diverse origins of the many cell types that contribute to the OFT. We also discuss the clinical implications of selected key findings for our understanding of the etiology of CHDs and particularly OFT malformations.

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