Detection and Sorting of Extracellular Vesicles and Viruses Using nanoFACS.
Authors: Morales-Kastresana A, Welsh JA, Jones JC
Abstract
Extracellular vesicles (EVs) are sub-micron-sized membranous spheres secreted by cells. EVs play a functional role as intercellular communicators and are associated with a number of diseases. Research into EVs is an area of growing interest due their many potential uses as therapeutic agents, as diagnostic and theranostic biomarkers, and as regulators of cellular biology. Flow cytometry is a popular method for enumerating and phenotyping EVs, even though the majority of EVs are below the detection sensitivity of most commercially available flow cyt...
Source: Current Protocols in Cytometry - December 18, 2020 Category: Molecular Biology Tags: Curr Protoc Cytom Source Type: research
Live Imaging of the Lung.
Authors: Brzoska T, Kaminski TW, Sundd P
Abstract
Live imaging is critical to determining the dynamics and spatial interactions of cells within the tissue environment. In the lung, this has proven to be difficult due to the motion brought about by ventilation and cardiac contractions. A previous version of this Current Protocols in Cytometry article reported protocols for imaging ex vivo live lung slices and the intact mouse lung. Here, we update those protocols by adding new methodologies, new approaches for quantitative image analysis, and new areas of potential application. © 2020 Wiley Periodicals LLC...
Source: Current Protocols in Cytometry - November 24, 2020 Category: Molecular Biology Tags: Curr Protoc Cytom Source Type: research
Small Particle Fluorescence and Light Scatter Calibration Using FCMPASS Software.
This article covers the workflow of implementing calibration using FCMPASS as follows: acquisition of fluorescence and light scatter calibration materials, cataloguing the reference materials for use in the software, creating cytometer databases and datasets to associate calibration data and fcs files, importing fcs files for calibration, inputting fluorescence calibration parameters, inputting light scatter calibration parameters, and applying the calibration to fcs files. Published 2020. U.S. Government. Basic Protocol 1: Acquisition and gating of light scatter calibration materials Basic Protocol 2: Acquisition and gati...
Source: Current Protocols in Cytometry - September 18, 2020 Category: Molecular Biology Tags: Curr Protoc Cytom Source Type: research
Optimized Stochastic Optical Reconstruction Microscopy for Imaging Chromatin Structure in Pathological Tissue.
Authors: Xu J, Ma H, Liu Y
Abstract
Direct visualization of higher-order chromatin structure at the molecular scale is of great importance for understanding the impact of chromatin organization on gene expression in many biological processes. Understanding the changes in chromatin structure during pathological processes requires the use of in vivo models and clinical samples, and formalin-fixed, paraffin-embedded (FFPE) tissue is the most widespread form of preservation. Here we describe the details of PathSTORM, an optimized stochastic optical reconstruction microscopy (STORM) protocol for high-quality su...
Source: Current Protocols in Cytometry - August 13, 2020 Category: Molecular Biology Tags: Curr Protoc Cytom Source Type: research
Flow Cytometric Quantification of Granulocytic Alkaline Phosphatase Activity in Unlysed Whole Blood.
Authors: Bardina J, Rico LG, Ward MD, Bradford JA, Juncà J, Petriz J
Abstract
Translational research has improved the diagnosis and follow-up of hematological diseases and malignancies. However, some classical diagnostics used for research and clinical practice that have remain practically unchanged for decades may be better addressed through advances in flow cytometry technology, whereby more precise measurements may be implemented in a straightforward manner. The current method for semiquantitative analysis of granulocytic alkaline phosphatase (GAP) activity is still based on observer-dependent color-in...
Source: Current Protocols in Cytometry - June 17, 2020 Category: Molecular Biology Tags: Curr Protoc Cytom Source Type: research
Practical Guidelines for Collection, Manipulation and Inactivation of SARS-CoV-2 and COVID-19 Clinical Specimens.
Authors: Bain W, Lee JS, Watson AM, Stitt-Fischer MS
Abstract
SARS-CoV-2 is a novel coronavirus that causes the acute respiratory disease-Coronavirus disease 2019 (COVID-19)-which has led to a global health crisis. Currently, no prophylactics or therapies exist to control virus spread or mitigate the disease. Thus, the risk of infection for physicians and scientists is high, requiring work to be conducted in Biosafety Level-3 (BSL-3) facilities if virus will be isolated or propagated. However, inactivation of the virus can enable safe handling at a reduced biosafety level, making samples accessible to a di...
Source: Current Protocols in Cytometry - June 7, 2020 Category: Molecular Biology Tags: Curr Protoc Cytom Source Type: research
Acquisition of High-Quality Spectral Flow Cytometry Data.
Authors: Fox A, Dutt TS, Karger B, Obregón-Henao A, Anderson GB, Henao-Tamayo M
Abstract
Flow cytometry allows the visualization of physical, functional, and/or biological properties of cells including antigens, cytokines, size, and complexity. With increasingly large flow cytometry panels able to analyze up to 50 parameters, there is a need to standardize flow cytometry protocols to achieve high-quality data that can be input into analysis algorithms. Without this clean data, algorithms may incorrectly categorize the cell populations present in the samples. In this protocol, we outline a comprehensive me...
Source: Current Protocols in Cytometry - May 19, 2020 Category: Molecular Biology Tags: Curr Protoc Cytom Source Type: research
Automated Motion Tracking and Data Extraction for Red Blood Cell Biomechanics.
We describe a workflow process using Image-Pro to identify and track red blood cells in a video, take measurements, and export the data for use in statistical analysis tools. The information in this protocol can be applied to large-scale blood studies where entire cell populations need to be analyzed from many cohorts of donors. © 2020 The Authors. Basic Protocol 1: Enhancing raw video for motion tracking Basic Protocol 2: Extracting motion tracking data from enhanced video.
PMID: 32391975 [PubMed - as supplied by publisher] (Source: Current Protocols in Cytometry)
Source: Current Protocols in Cytometry - May 12, 2020 Category: Molecular Biology Tags: Curr Protoc Cytom Source Type: research
Acute Myeloid Leukemia Minimal Residual Disease Detection: The Difference from Normal Approach.
Authors: Wood BL
Abstract
The identification of residual leukemia following therapy, termed minimal or measurable residual disease (MRD), has emerged as one of the most important prognostic factors for patients with acute leukemia, including acute myeloid leukemia (AML). Flow cytometry is a preferred method for MRD detection due to its general applicability and the rapid results that it makes available. In this article, the basic protocol outlines a simple and efficient method for the labeling of hematopoietic cells from bone marrow or peripheral blood with a panel of monoclonal antibodies designed both to...
Source: Current Protocols in Cytometry - April 22, 2020 Category: Molecular Biology Tags: Curr Protoc Cytom Source Type: research
Estimation of Microbial Viability Using Flow Cytometry.
This article provides a commentary and several protocols have been included to ensure that users have a firm basis for attempting these reasonably difficult assays on traditional flow cytometer instruments. What is clear is that each assay must be carefully validated with the particular microorganism of interest before being applied in any research, clinical, or service form. © 2020 The Authors.
PMID: 32289207 [PubMed - as supplied by publisher] (Source: Current Protocols in Cytometry)
Source: Current Protocols in Cytometry - April 15, 2020 Category: Molecular Biology Tags: Curr Protoc Cytom Source Type: research
Training Novices in Generation and Analysis of High-Dimensional Human Cell Phospho-Flow Cytometry Data.
This article presents a single experiment designed to introduce a trainee to multiple advanced bench and analysis techniques, including high-dimensional cytometry, profiling cell signaling networks, functional assays with primary human tissue, and single-cell analysis with machine learning tools. The trainee is expected to have only minimal laboratory experience and is not required to have any prior training in flow cytometry, immunology, or data science. This article aims to introduce the advanced research areas with a design that is robust enough that novice trainees will succeed, flexible enough to allow some project cu...
Source: Current Protocols in Cytometry - April 8, 2020 Category: Molecular Biology Tags: Curr Protoc Cytom Source Type: research
Panel Design and Optimization for High-Dimensional Immunophenotyping Assays Using Spectral Flow Cytometry.
Authors: Ferrer-Font L, Pellefigues C, Mayer JU, Small SJ, Jaimes MC, Price KM
Abstract
Technological advances in fluorescence flow cytometry and an ever-expanding understanding of the complexity of the immune system have led to the development of large (20+ parameters) flow cytometry panels. However, as panel complexity and size increase, so does the difficulty involved in designing a high-quality panel, accessing the instrumentation capable of accommodating large numbers of parameters, and analyzing such high-dimensional data. A recent advancement is spectral flow cytometry, which in contrast to conventi...
Source: Current Protocols in Cytometry - March 10, 2020 Category: Molecular Biology Tags: Curr Protoc Cytom Source Type: research
Single-Cell Analysis of Cytokine mRNA and Protein Expression by Flow Cytometry.
Authors: Pal R, Schaubhut J, Clark D, Brown L, Stewart JJ
Abstract
Understanding how immune cells respond to external stimuli such as pathogens or drugs is a key component of biomedical research. Critical to the immune response are the expression of cell-surface receptors and the secretion of cytokines, which are tightly regulated by gene expression and protein synthesis. Previously, cytokine mRNA expression levels have been measured from bulk analysis of heterogeneous or sorted cell populations, and the correlation between cytokine mRNA expression and protein levels using these techniques can be highly va...
Source: Current Protocols in Cytometry - February 25, 2020 Category: Molecular Biology Tags: Curr Protoc Cytom Source Type: research
Enumeration of Fetal Red Blood Cells, Hemoglobin-Specific RBC Cells, and F Reticulocytes in Human Blood.
This article presents three applications of red blood cell (RBC) analysis-quantitation of fetal red cells, F-cell enumeration, and F-reticulocyte analysis-which improve diagnostic precision, sensitivity, and specificity, and provide better laboratory indicators of therapeutic efficacy in a variety of hematologic and obstetric disorders. Such advances also include the measurement and quantitation of RBC hemoglobins and their relative ribonucleic acid levels. These advances not only promise to improve diagnostic accuracy and laboratory precision over techniques such as the traditional manual reticulocyte counting method and ...
Source: Current Protocols in Cytometry - January 5, 2020 Category: Molecular Biology Tags: Curr Protoc Cytom Source Type: research
Cell Volume Measurements by Optical Transmission Microscopy.
This article describes a method for measurement of the volumes of adherent cells using a standard light microscope. A coverslip with attached cells is placed in a shallow chamber in a medium containing a strongly absorbing and cell-impermeant dye, Acid Blue 9. When such a sample is imaged in transmitted light at a wavelength of maximum dye absorption (630 nm), the resulting contrast quantitatively reflects cell thickness; once the thickness is known at every point, the volume can be computed as well. Technical details, interpretation of data, and possible artifacts are discussed. © 2019 by John Wiley & Sons, Inc.
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Source: Current Protocols in Cytometry - January 5, 2020 Category: Molecular Biology Tags: Curr Protoc Cytom Source Type: research
