Confocal Microscopy: Principles and Modern Practices.
This article provides a concise introduction to confocal microscopy. © 2019 by John Wiley & Sons, Inc.
PMID: 31876974 [PubMed - in process] (Source: Current Protocols in Cytometry)
Source: Current Protocols in Cytometry - December 27, 2019 Category: Molecular Biology Tags: Curr Protoc Cytom Source Type: research
Basics of Expansion Microscopy.
Authors: Klimas A, Gallagher B, Zhao Y
Abstract
Optical imaging techniques are often used in neuroscience to understand brain function and discern disease pathogenesis. However, the optical diffraction limit precludes conventional optical imaging approaches from resolving nanoscopic structures with feature sizes smaller than 300 nm. Expansion microscopy (ExM) circumvents this limit by physically expanding preserved tissues embedded in a swellable hydrogel. Biomolecules of interest are covalently linked to a polymer matrix, which is then isotropically expanded at least 100-fold in size in pure water after m...
Source: Current Protocols in Cytometry - November 26, 2019 Category: Molecular Biology Tags: Curr Protoc Cytom Source Type: research
Simultaneous Polychromatic Immunofluorescent Staining of Tissue Sections and Consecutive Imaging of up to Seven Parameters by Standard Confocal Microscopy.
Authors: Schmidt AJ, Mayer JU, Wallace PK, Ronchese F, Price KM
Abstract
Confocal microscopy has been an important imaging tool for life scientists for over 20 years. Early techniques focused on indirect staining processes that involved staining with an unconjugated primary antibody, followed by incubation with a secondary fluorescent antibody that would reveal and amplify the signal of the primary antibody. With more and more directly conjugated fluorescent primary antibodies becoming commercially available, staining with multiple fluorescent primary antibodies is now more frequent. To date, staining with...
Source: Current Protocols in Cytometry - November 26, 2019 Category: Molecular Biology Tags: Curr Protoc Cytom Source Type: research
Assessment of DNA Susceptibility to Denaturation as a Marker of Chromatin Structure.
This article presents a flow cytometric procedure for assessing DNA denaturation based on application of the metachromatic property of acridine orange (AO) to differentially stain single- versus double-stranded DNA. This approach circumvents limitations of biochemical methods of examining DNA denaturation, in particular the fact that the latter destroy higher orders of chromatin structure and that, being applied to bulk cell populations, they cannot detect heterogeneity of individual cells. Because the metachromatic properties of AO have also found application in other cytometric procedures, such as differential staining o...
Source: Current Protocols in Cytometry - November 26, 2019 Category: Molecular Biology Tags: Curr Protoc Cytom Source Type: research
Immunophenotypic Detection of Measurable Residual (Stem Cell) Disease Using LAIP Approach in Acute Myeloid Leukemia.
Authors: Zeijlemaker W, Kelder A, Cloos J, Schuurhuis GJ
Abstract
Half of the patients with acute myeloid leukemia (AML), who achieve complete remission after chemotherapy treatment, will ultimately experience a relapse. Measurable residual disease (MRD) is an important post-treatment risk factor in AML, because it gives additional information about the depth of the remission. Within MRD, the small population of leukemic stem cells (LSCs) is thought to be at the base of the actual relapse. In this protocol, the flow cytometric detection of MRD and LSCs herein is outlined. We give a detailed overview of the...
Source: Current Protocols in Cytometry - November 26, 2019 Category: Molecular Biology Tags: Curr Protoc Cytom Source Type: research
Monitoring of Measurable Residual Disease in Multiple Myeloma by Multiparametric Flow Cytometry.
Authors: Soh KT, Wallace PK
Abstract
Recent interest in high sensitivity multiple myeloma (MM) measurable residual disease (MRD) testing is a direct consequence of the high-quality responses achieved using novel therapeutic agents and better treatment strategies. Traditional diagnostic measures such as immunohistochemistry and morphology have detection sensitivities of only 10-2 - 10-3, which do not reliably predict progression free survival (PFS) or overall survival (OS) after these treatments. Contemporary monitoring of MM MRD has switched to more sensitive platforms such as quantitative allele-specific ...
Source: Current Protocols in Cytometry - October 16, 2019 Category: Molecular Biology Tags: Curr Protoc Cytom Source Type: research
Detection of Histone H2AX Phosphorylation on Ser-139 as an Indicator of DNA Damage.
Authors: Zhao H, Huang X, Halicka HD, Darzynkiewicz Z
Abstract
This unit describes immunocytochemical detection of histone H2AX phosphorylated on Ser-139 (γH2AX) to reveal DNA damage, particularly when the damage involves the presence of DNA double-strand breaks (DSBs). These breaks often result from DNA damage induced by ionizing radiation or by treatment with anticancer drugs such as DNA topoisomerase inhibitors. Furthermore, DSBs are generated in the course of DNA fragmentation during apoptosis. The unit presents strategies to distinguish radiation- or drug-induced DNA breaks from those intrinsically f...
Source: Current Protocols in Cytometry - June 28, 2019 Category: Molecular Biology Tags: Curr Protoc Cytom Source Type: research
Flow Cytometry Analysis of Free Intracellular NAD+ Using a Targeted Biosensor.
Authors: Eller JM, Stewart ML, Slepian AJ, Markwardt S, Wiedrick J, Cohen MS, Goodman RH, Cambronne XA
Abstract
Flow cytometry approaches combined with a genetically encoded targeted fluorescent biosensor are used to determine the subcellular compartmental availability of the oxidized form of nicotinamide adenine dinucleotide (NAD+ ). The availability of free NAD+ can affect the activities of NAD+ -consuming enzymes such as sirtuin, PARP/ARTD, and cyclic ADPR-hydrolase family members. Many methods for measuring the NAD+ available to these enzymes are limited because they cannot determine free NAD+ as it ex...
Source: Current Protocols in Cytometry - December 19, 2018 Category: Molecular Biology Tags: Curr Protoc Cytom Source Type: research
Flow Cytometry Instrumentation - An Overview.
Authors: Büscher M
Abstract
The term flow cytometry, used since the seventies, describes a technology employed mainly in biology and medicine to measure and classify suspended particles, e.g., cells or microspheres. Measurable cell parameters include: geometric properties, such as cell size (diameter, surface area, volume); physiological properties (membrane potential, integrity, vitality); and quantities of DNA, RNA, cytokines, surface antigens, nuclear antigens, enzymes, and proteins.
PMID: 30444583 [PubMed - as supplied by publisher] (Source: Current Protocols in Cytometry)
Source: Current Protocols in Cytometry - November 19, 2018 Category: Molecular Biology Tags: Curr Protoc Cytom Source Type: research
Flow Cytometry Method Validation Protocols.
Authors: Selliah N, Eck S, Green C, Oldaker T, Stewart J, Vitaliti A, Litwin V
Abstract
Analytical method validation provides a means to ensure that data are credible and reproducible. This unit will provide a brief introduction to analytical method validation as applied to cellular analysis by flow cytometry. In addition, the unit will provide practical procedures for three different types of validation. The first is a limited validation protocol that is applicable for research settings and non-regulated laboratories. The second is validation protocol that presents the minimum validation requirements in r...
Source: Current Protocols in Cytometry - November 13, 2018 Category: Molecular Biology Tags: Curr Protoc Cytom Source Type: research
Multiphoton Imaging of Collagen, Elastin, and Calcification in Intact Soft-Tissue Samples.
Authors: Gade PS, Robertson AM, Chuang CY
Abstract
Multiphoton-induced second-harmonic generation and two-photon excitation enable imaging of collagen and elastin fibers at micron-level resolution to depths of hundreds of microns, without the use of exogenous stains. These attributes can be leveraged for quantitative analysis of the 3D architecture of collagen and elastin fibers within intact, soft tissue specimens such as the artery and bladder wall. This architecture influences the function of intramural cells and also plays a primary role in determining tissue passive mechanical properties. Calcificat...
Source: Current Protocols in Cytometry - November 2, 2018 Category: Molecular Biology Tags: Curr Protoc Cytom Source Type: research
Cell Cycle Analysis of Hematopoietic Stem and Progenitor Cells by Multicolor Flow Cytometry.
Authors: Galvin A, Weglarz M, Folz-Donahue K, Handley M, Baum M, Mazzola M, Litwa H, Scadden DT, Silberstein L
Abstract
Maintenance of hematopoietic stem cell (HSC) quiescence is critical for self-renewal and differentiation into mature lineages. Therefore, the ability to reliably detect abnormal HSC cycling is essential for experiments that seek to investigate abnormalities of HSC function. The ability to reproducibly evaluate cell cycle status in a rare cell subset requires careful optimization of multiple parameters during cell preparation and sample processing. Here, we describe a method where data acq...
Source: Current Protocols in Cytometry - October 20, 2018 Category: Molecular Biology Tags: Curr Protoc Cytom Source Type: research
Flow Cytometric Monitoring for Residual Disease in B Lymphoblastic Leukemia Post T Cell Engaging Targeted Therapies.
We describe herein a strategy for flow cytometric monitoring for residual disease in patients with B-LL post T cell-engaging anti-CD19 and anti-CD22 therapies. © 2018 by John Wiley & Sons, Inc.
PMID: 30212602 [PubMed - as supplied by publisher] (Source: Current Protocols in Cytometry)
Source: Current Protocols in Cytometry - September 15, 2018 Category: Molecular Biology Tags: Curr Protoc Cytom Source Type: research
Deriving Extracellular Vesicle Size From Scatter Intensities Measured by Flow Cytometry.
Authors: de Rond L, Coumans FAW, Nieuwland R, van Leeuwen TG, van der Pol E
Abstract
Flow cytometry is commonly used to investigate the potential for extracellular vesicles (EVs) to be biomarkers of disease. A typical flow cytometer detects fluorescence and scatter intensities of single EVs in arbitrary units. These arbitrary units complicate data interpretation and data comparison between different flow cytometers. For example, comparison of detected EV concentrations requires knowledge of the detectable EV sizes. Using Mie theory and knowledge of the optical configuration of the flow cytometer, EV size c...
Source: Current Protocols in Cytometry - September 2, 2018 Category: Molecular Biology Tags: Curr Protoc Cytom Source Type: research
A Practical Cryopreservation and Staining Protocol for Immunophenotyping in Population Studies.
Authors: Barcelo H, Faul J, Crimmins E, Thyagarajan B
Abstract
Large population-based cohort studies, through their prospective collection of a broad range of health information, represent an invaluable resource for novel insights into the pathogenesis of human diseases. Collection and cryopreservation of viable cells from blood samples is becoming increasingly common in large cohorts as these cells are a valuable resource for immunophenotyping and functional studies. The cryopreservation of peripheral blood mononuclear cells (PBMCs), thawing, and immunophenotyping protocols used to immunophenotype 9938 pa...
Source: Current Protocols in Cytometry - July 25, 2018 Category: Molecular Biology Tags: Curr Protoc Cytom Source Type: research
