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12 ARTICLES PUBLISHED IN JoVE

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Neuroscience

An Optic Nerve Crush Injury Murine Model to Study Retinal Ganglion Cell Survival
Zhongshu Tang 1, Shuihua Zhang 1,2, Chunsik Lee 1, Anil Kumar 1, Pachiappan Arjunan 1, Yang Li 1, Fan Zhang 1, Xuri Li 1
1National Eye Institute, NIH, 2Ophthalmology Department, The Second Hospital of Harbin Medical University

This protocol shows how to retrogradely label retinal ganglion cells, and how to subsequently make an optic nerve crush injury in order to analyze retinal ganglion cell survival and apoptosis. It is an experimental disease model for different types of optic neuropathy, including glaucoma.

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Medicine

A Mouse Model of the Cornea Pocket Assay for Angiogenesis Study
Zhongshu Tang 1, Fan Zhang 1, Yang Li 1, Pachiappan Arjunan 1, Anil Kumar 1, Chunsik Lee 1, Xuri Li 1
1National Eye Institute

The cornea is unique in that it lacks vascular tissues. However, robust blood vessel growth and survival can be induced in the cornea by potent angiogenic factors. Therefore, the cornea can provide with us a valuable tool for angiogenic studies. This protocol demonstrates how to perform the mouse model of cornea pocket assay and how to assess the angiogenesis induced by angiogenic factors using this model.

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Biology

Conversion of a Capture ELISA to a Luminex xMAP Assay using a Multiplex Antibody Screening Method
Harold N. Baker 1, Robin Murphy 1, Erica Lopez 1, Carlos Garcia 1,2
1Chemistry Research and Development, Luminex Corporation, 2Global Marketing, Luminex Corporation

An ELISA can be easily converted to a Luminex xMAP assay and, through the benefits of multiplexing, several antibodies can be screened simultaneously to identify an optimum antibody pair, resulting in increased sensitivity and dynamic range, while reducing assay cost.

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Bioengineering

Imaging Denatured Collagen Strands In vivo and Ex vivo via Photo-triggered Hybridization of Caged Collagen Mimetic Peptides
Yang Li 1, Catherine A. Foss 2, Martin G. Pomper 2,3, S. Michael Yu 1,3
1Department of Bioengineering, University of Utah, 2Department of Radiology and Radiological Science, Johns Hopkins University School of Medicine, 3Institute for NanoBiotechnology, Johns Hopkins University

This procedure demonstrates in vivo near IR fluorescence imaging of collagen remodeling activities in mice as well as ex vivo staining of collagens in tissue sections using caged collagen mimetic peptides that can be photo-triggered to hybridize with denatured collagen strands.

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Immunology and Infection

Intravital Microscopy Imaging of the Liver following Leishmania Infection: An Assessment of Hepatic Hemodynamics
Sreekanth Dasari 1, Pascal Weber 1, Camélia Makhloufi 1, Erica Lopez 2, Claire-Lise Forestier 1
1INSERM 1095, URMITE, University of Aix-Marseille, 2U.F.R de Médecine, University of Aix-Marseille

This article reports on a detailed method for the dynamic measurement and quantification of blood flow velocity within individual blood vessels of the mouse liver vasculature using intravital microscopy imaging in combination with a specific methodology for image acquisition and analysis.

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Biology

Assessing Autophagic Flux by Measuring LC3, p62, and LAMP1 Co-localization Using Multispectral Imaging Flow Cytometry
Haley R. Pugsley 1
1Amnis Biology Department, MilliporeSigma

Here, multispectral imaging flow cytometry with an analytical feature that compares bright detail images of 3 autophagy markers and quantifies their co-localization, along with LC3 spot counting, was used to measure autophagy in an objective, quantitative, and statistically robust manner.

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JoVE Journal

Differential Effects of Lipid-lowering Drugs in Modulating Morphology of Cholesterol Particles
Shanmugavel Madasamy 1, David Liu 1, Jason Lundry 1, Benjamin Alderete 2, Raymond Kong 2, J. Paul Robinson 3, Alan H.B. Wu 1,4, Edward P. Amento 5
1Plaxgen Inc, 2Millipore Sigma, 3Cytometry Laboratories, Purdue University, 4San Francisco General Hospital, 2M16 Clinical Chemistry, University of California, San Francisco, 5Molecular Medicine Research Institute

The objective of this study was to evaluate in vitro lipid-lowering drug effects in modulating the morphology of cholesterol particles. Comparison of lipid-lowering drugs revealed variations in their effect in modulating the morphological features of cholesterol particles.

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Bioengineering

An Automated Method to Perform The In Vitro Micronucleus Assay using Multispectral Imaging Flow Cytometry
Matthew A. Rodrigues 1
1Biology Department, Luminex Corporation

The in vitro micronucleus assay is a well-established method for evaluating genotoxicity and cytotoxicity but scoring the assay using manual microscopy is laborious and suffers from subjectivity and inter-scorer variability. This paper describes the protocol developed to perform a fully automated version of the assay using multispectral imaging flow cytometry.

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Immunology and Infection

A Rapid, Multiplex Dual Reporter IgG and IgM SARS-CoV-2 Neutralization Assay for a Multiplexed Bead-Based Flow Analysis System
Stephen Angeloni 1, Andrew Cameron 2, Nicole D. Pecora 2,3, Sherry Dunbar 1
1Luminex Corporation, 2Departments of Pathology and Laboratory Medicine, Clinical Microbiology, University of Rochester Medical Center, 3Department of Microbiology and Immunology, University of Rochester Medical Center

A flow analysis system for bead-based multiplexed assays which provides a two-reporter readout was used for the development of multiplex serological and antibody neutralization assays that can simultaneously measure neutralizing IgG and IgM antibodies for SARS-CoV-2.

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Medicine

Isolation of Monocyte-Macrophage Lineage Cells from Rat Bones by Secondary Adherence Method
Xiaoli Jin *1, Yang Li *2, Xuanwei Chen 1, Jin Chen 1, Jian Xu 1
1School of Medical Technology and Information Engineering, Zhejiang Chinese Medical University, 2School of Basic Medical Sciences, Fudan University

Here we present a protocol for the isolation of BMMs from SD rats, called the secondary adherence method.

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Immunology and Infection

Efficient Transfection of In vitro Transcribed mRNA in Cultured Cells Using Peptide-Poloxamine Nanoparticles
Qin Xiao *1, Yuheng Liu *1, Dandan Zhang 1, Chao Li 1, Qihua Yang 1, Dongshui Lu 1, Weijun Zhang 1, Joseph Rosenecker 2, Quanming Zou 1, Yang Li 3, Shan Guan 1
1National Engineering Research Center of Immunological Products, Department of Microbiology and Biochemical Pharmacy, Third Military Medical University, 2Department of Pediatrics, Ludwig-Maximilians University of Munich, 3Department of Pharmacy, Southwest Hospital, Third Military Medical University

A self-assembled peptide-poloxamine nanoparticle (PP-sNp) is developed using a microfluidic mixing device to encapsulate and deliver in vitro transcribed messenger RNA. The described mRNA/PP-sNp could efficiently transfect cultured cells in vitro.

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Bioengineering

Automation of the Micronucleus Assay Using Imaging Flow Cytometry and Artificial Intelligence
Matthew A. Rodrigues 1, María Gracia García Mendoza 1, Raymond Kong 1, Alexandra Sutton 1, Haley R. Pugsley 1, Yang Li 1, Brian E. Hall 1, Darin Fogg 1, Lars Ohl 1, Vidya Venkatachalam 1
1Amnis Flow Cytometry, Luminex Corporation

The micronucleus (MN) assay is a well-established test for quantifying DNA damage. However, scoring the assay using conventional techniques such as manual microscopy or feature-based image analysis is laborious and challenging. This paper describes the methodology to develop an artificial intelligence model to score the MN assay using imaging flow cytometry data.

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