1Institute of Bio- and Geosciences, IBG-1: Biotechnology, Forschungszentrum Juelich GmbH
In this protocol the fabrication, setup and basic operation of a microfluidic picoliter bioreactor (PLBR) for single-cell analysis of prokaryotic microorganisms is introduced. Industrially relevant microorganisms were analyzed as proof of principle allowing insights into growth rate, morphology, and phenotypic heterogeneity over certain time periods, hardly possible with conventional methods.
Published December 6, 2013. Keywords: Bioengineering, Soft lithography, SU-8 lithography, Picoliter bioreactor, Single-cell analysis, Polydimethylsiloxane, Corynebacterium glutamicum, Escherichia coli, Microfluidics, Lab-on-a-chip
1Department of Chemical Engineering, Massachusetts Institute of Technology
We present a simple protocol to obtain fluorescence microscopy movies of growing yeast cells, and a GUI-based software package to extract single-cell time series data. The analysis includes automated lineage and division time assignment integrated with visual inspection and manual curation of tracked data.
Published July 18, 2013. Keywords: Microbiology, Cellular Biology, Molecular Biology, Genetics, Biophysics, Saccharomyces cerevisiae, Microscopy, Fluorescence, Cell Biology, microscopy/fluorescence and time-lapse, budding yeast, gene expression dynamics, segmentation, lineage tracking, image tracking, software, yeast, cells, imaging
JoVE Immunology and Infection
1Molecular Genetics Group, Groningen Biomolecular Sciences and Biotechnology Institute, Centre for Synthetic Biology, University of Groningen
This protocol provides a step-by-step procedure to monitor single cell behavior of different bacteria in time using automated fluorescence time-lapse microscopy. Furthermore, we provide guidelines how to analyze the microscopy images.
Published July 28, 2011. Keywords: Immunology, time-lapse fluorescence microscopy, single cell analysis, cell history, cell growth, development, promoter activity, protein localization, GFP, Bacillus subtilis, Streptococcus pneumoniae
1Bredesen Center, University of Tennessee, Knoxville, 2Center for Nanophase Materials Sciences, Oak Ridge National Laboratory, 3Department of Materials Science and Engineering, University of Tennessee, Knoxville
A microfabricated device with sealable femtoliter-volume reaction chambers is described. This report includes a protocol for sealing cell-free protein synthesis reactants inside these chambers for the purpose of understanding the role of crowding and confinement in gene expression.
Published March 11, 2015. Keywords: Bioengineering, Cell-free, synthetic biology, microfluidics, noise biology, soft lithography, femtoliter volumes
1Division of Hematology/Oncology, Department of Oncology, East Carolina University
Commonly used, highly accessible methods for examining cell migration and invasion in vitro are described. The first method is the cell wound closure assay that measures cell motility. The second method is the transwell migration and invasion assay that assesses the chemotactic and invasive capacity of cells.
Published June 1, 2014. Keywords: Bioengineering, Cell migration, cell invasion, chemotaxis, transwell assay, wound closure assay, time-lapse microscopy
1Department of Cell Biology, Harvard Medical School
The mesothelial clearance assay described here takes advantage of fluorescently labeled cells and time-lapse video microscopy to visualize and quantitatively measure the interactions of ovarian cancer multicellular spheroids and mesothelial cell monolayers. This assay models the early steps of ovarian cancer metastasis.
Published February 17, 2012. Keywords: Medicine, Ovarian Cancer, Metastasis, In vitro Model, Mesothelial, Spheroid
1Department of Molecular Biosciences, Rice Institute for Biomedical Research, Northwestern University
Prion-like propagation of protein aggregates has recently emerged as being implicated in many neurodegenerative diseases. The goal of this protocol is to describe, how to use the nematode C. elegans as a model system to monitor protein spreading and to investigate prion-like phenomena.
Published January 8, 2015. Keywords: Cellular Biology, Caenorhabditis elegans, neurodegenerative diseases, protein misfolding diseases, prion-like spreading, cell-to-cell transmission, protein aggregation, non-cell autonomous toxicity, proteostasis
JoVE Immunology and Infection
1Medical Microbiology, University Medical Center Utrecht
We present methods to study the effect of PSMs and other toxins secreted by Staphylococcus aureus on neutrophils using flow cytometry and fluorescence microscopy.
Published July 17, 2013. Keywords: Infection, Immunology, Cellular Biology, Infectious Diseases, Microbiology, Genetics, Medicine, Biomedical Engineering, Bioengineering, Neutrophils, Staphylococcus aureus, Bacterial Toxins, Microscopy, Fluorescence, Time-Lapse Imaging, Phagocytosis, phenol soluble modulins, PSMs, Polymorphonuclear Neutrophils, PMNs, intracellular expression, time-lapse microscopy, flow cytometry, cell, isolation, cell culture
JoVE Immunology and Infection
1Department of Chemical and Biomolecular Engineering, University of Houston, 2Division of Pediatrics, Research Unit 907, University of Texas MD Anderson Cancer Center
We describe a single-cell high-throughput assay to measure cytotoxicity of T cells when incubated with tumor target cells. This method employs a dense, elastomeric array of sub-nanoliter wells (~100,000 wells/array) to spatially confine the T cells and target cells at defined ratios and is coupled to fluorescence microscopy to monitor effector-target conjugation and subsequent apoptosis.
Published February 2, 2013. Keywords: Cancer Biology, Immunology, Cellular Biology, Molecular Biology, Medicine, Chemical Engineering, Biomolecular Engineering, Bioengineering, Immunotherapy, Adoptive, Microfluidics, Nanowell arrays, PDMS, BioStation, T Cells, tumor target cells, labeling, cytotoxicity, microscopy, assay
1Department of Oncological Sciences, Huntsman Cancer Institute, University of Utah, 2Fluorescence Microscopy Core Facility, University of Utah
Here we describe a basic protocol to image and quantify the mitotic timing of live mammalian tissue culture cells after siRNA transfection.
Published June 6, 2010. Keywords: Cellular Biology, microscopy, live imaging, mitosis, transfection, siRNA
1School of Biosciences, University of Birmingham, 2School of Medicine, University of Birmingham, 3Centre for Human Reproductive Science, Birmingham Women’s Hospital
Stimulus-evoked [Ca2+]i signals of individual human sperm are assessed. Motile cells are loaded with Ca2+-sensitive fluorescent dye (AM-ester method) and immobilised in a perfusable chamber. Cells are imaged by time-lapse fluorescence microscopy and stimulated via the perfusing medium. Responses of single cells (or regions) are analysed offline using Excel.
Published June 16, 2010. Keywords: Cellular Biology, sperm, human, calcium, fluorescence microscopy