Attenuation of replication stress-induced premature cellular senescence to assess anti-aging modalities.
Authors: Zhao H, Darzynkiewicz Z
Abstract
Described is an in vitro model of premature senescence in pulmonary adenocarcinoma A549 cells induced by persistent DNA replication stress in response to treatment with the DNA damaging drug mitoxantrone (Mxt). The degree of cellular senescence, based on characteristic changes in cell morphology, is measured by laser scanning cytometry. Specifically, the flattening of cells grown on slides (considered the hallmark of cellular senescence) is measured as the decline in local intensity of DNA-associated DAPI fluorescence (represented by maximal pixels). This change is...
Source: Current Protocols in Cytometry - November 20, 2015 Category: Molecular Biology Tags: Curr Protoc Cytom Source Type: research
Correlative fluorescence and electron microscopy.
Authors: Schirra RT, Zhang P
Abstract
Correlative fluorescence and electron microscopy (CFEM) is a multimodal technique that combines dynamic and localization information from fluorescence methods with ultrastructural data from electron microscopy, to give new information about how cellular components change relative to the spatiotemporal dynamics within their environment. In this review, we will discuss some of the basic techniques and tools of the trade for utilizing this attractive research method, which is becoming a very powerful tool for biology labs. The information obtained from correlative methods...
Source: Current Protocols in Cytometry - November 20, 2015 Category: Molecular Biology Tags: Curr Protoc Cytom Source Type: research
FRET Imaging by Laser Scanning Cytometry on Large Populations of Adherent Cells.
Authors: Doan-Xuan QM, Szalóki N, Tóth K, Szöllősi J, Bacso Z, Vámosi G
Abstract
The application of FRET (fluorescence resonance energy transfer) sensors for monitoring protein-protein interactions under vital conditions is attracting increasing attention in molecular and cell biology. Laser-scanning cytometry (LSC), a slide-based sister procedure to flow cytometry, provides an opportunity to analyze large populations of adherent cells or 2-D solid tissues in their undisturbed physiological settings. Here we provide an LSC-based three-laser protocol for high-throughput ratiometric FRET measurements ut...
Source: Current Protocols in Cytometry - November 20, 2015 Category: Molecular Biology Tags: Curr Protoc Cytom Source Type: research
High-resolution cytometry for high-content cell cycle analysis.
Authors: Furia L, Pelicci P, Faretta M
Abstract
One of the major limitations of flow cytometry (FCM) is the absence of an intracellular view. Automated microscopy and image analysis, together with technological developments, led to new approaches in cytometry that bypass the above limitation, introducing high resolution, high content, and large statistical sampling. However, few attempts have been made, until now, to translate the wide repertoire of FCM assays into high-content image screening. This unit describes the implementation of an acquisition and analysis protocol for evaluation of the cell cycle b...
Source: Current Protocols in Cytometry - November 20, 2015 Category: Molecular Biology Tags: Curr Protoc Cytom Source Type: research
Confocal microscopy for high-resolution and high-content analysis of the cell cycle.
Authors: Furia L, Pelicci P, Faretta M
Abstract
Optical fluorescence microscopy offers a wide range of technological solutions to address many questions in biomedical research. Spatial resolution has been greatly improved by the use of confocal microscopes, providing a 3-D analysis of the intracellular space. Automation has contributed to make confocal analysis available for high-content image cytometry studies. However, the storage, browsing, and analysis of the amount of data generated can challenge the feasibility of such studies. Presented in this chapter is a multistep acquisition and analysis protoco...
Source: Current Protocols in Cytometry - November 20, 2015 Category: Molecular Biology Tags: Curr Protoc Cytom Source Type: research
Digital data acquisition and processing.
Authors: Naivar MA, Galbraith DW
Abstract
A flow cytometer is made up of many different subsystems that work together to measure the optical properties of individual cells within a sample. The data acquisition system (also called the data system) is one of these subsystems, and it is responsible for converting the electrical signals from the optical detectors into list-mode data. This unit describes the inner workings of the data system, and provides insight into how the instrument functions as a whole. Some of the information provided in this unit is applicable to everyday use of these instruments, and, a...
Source: Current Protocols in Cytometry - November 20, 2015 Category: Molecular Biology Tags: Curr Protoc Cytom Source Type: research
Light Sheet Fluorescence Microscopy (LSFM).
Authors: Adams MW, Loftus AF, Dunn SE, Joens MS, Fitzpatrick JA
Abstract
The development of confocal microscopy techniques introduced the ability to optically section fluorescent samples in the axial dimension, perpendicular to the image plane. These approaches, via the placement of a pinhole in the conjugate image plane, provided superior resolution in the axial (z) dimension resulting in nearly isotropic optical sections. However, increased axial resolution, via pinhole optics, comes at the cost of both speed and excitation efficiency. Light sheet fluorescent microscopy (LSFM), a century-old idea made po...
Source: Current Protocols in Cytometry - November 20, 2015 Category: Molecular Biology Tags: Curr Protoc Cytom Source Type: research
Semi-automated object tracking methods in biological imaging.
Authors: Davis MA, Praěský O, Sysko LR
Abstract
Time-lapse imaging is a rich data source offering potential kinetic information of cellular activity and behavior. Tracking and extracting measurements of objects from time-lapse datasets are challenges that result from the complexity and dynamics of each object's motion and intensity or the appearance of new objects in the field of view. A wide range of strategies for proper data sampling, object detection, image analysis, and post-analysis interpretation are available. Theory and methods for single-particle tracking, spot detection, and object linking are...
Source: Current Protocols in Cytometry - November 20, 2015 Category: Molecular Biology Tags: Curr Protoc Cytom Source Type: research
Quantification of Th1 and Th17 Cells with Intracellular Staining Following PMA/Ionomycin Stimulation.
Authors: Karlsson F, Hassan-Zahraee M
Abstract
Cytokine-producing cells are at the center of the adaptive immune responses, and quantifying these cells is an important aspect to build understanding of the immune response. In particular, Th1 and Th17 cells have been implicated in the pathogenesis of such diseases as inflammatory bowel disease, rheumatoid arthritis, and multiple sclerosis. Quantification of Th1 and Th17 cells can provide important information in research of these diseases and other Th1- and Th17-mediated immune disorders. In vitro stimulation of cells followed by surface and intracellular st...
Source: Current Protocols in Cytometry - November 20, 2015 Category: Molecular Biology Tags: Curr Protoc Cytom Source Type: research
Whole blood measurement of histone modifications linked to the epigenetic regulation of gene expression.
Authors: Watson M, Hedley D
Abstract
Rapid progress is being made to understand the regulatory mechanisms that underlie the epigenetic control of gene expression through histone modification. It is now recognized that this plays a major role in normal development and disease. This unit describes the application of flow cytometry to the study of epigenetic mechanisms by combining labeling of individual histone modifications and phenotypic markers, and it also discusses practical issues to optimize staining. The focus is on normal blood and samples from leukemia patients, but it can also be applied to cells ...
Source: Current Protocols in Cytometry - November 20, 2015 Category: Molecular Biology Tags: Curr Protoc Cytom Source Type: research
Application of click chemistry conditions for 5-bromo-2'-deoxyuridine determination through fenton and related reactions.
Authors: Cappella P, Pulici M, Gasparri F
Abstract
Mixtures of ascorbate and copper used in certain click chemistry experimental conditions act as oxidizing agents, catalyzing the formation of reactive oxygen species through Fenton and related reactions. Hydroxyl radicals act as chemical nucleases, introducing DNA strand breaks that can be exploited for BrdU immunostaining in place of acid denaturation. This procedure is readily applicable to high content analysis and flow cytometry assays, and provides results comparable to click chemistry EdU cycloaddition and classical BrdU immunodetection. Importantly,...
Source: Current Protocols in Cytometry - November 20, 2015 Category: Molecular Biology Tags: Curr Protoc Cytom Source Type: research
Cell volume measurements by optical transmission microscopy.
Authors: A Model M
Abstract
Cell volume is an important parameter in cell adaptation to anisosmotic stress, in the development of apoptosis and necrosis, and in the pathogenesis of several diseases. This unit describes a method for measuring the volume 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 thickne...
Source: Current Protocols in Cytometry - November 20, 2015 Category: Molecular Biology Tags: Curr Protoc Cytom Source Type: research
High-Sensitivity Detection of PNH Red Blood Cells, Red Cell Precursors, and White Blood Cells.
Authors: Sutherland DR, Illingworth A, Keeney M, Richards SJ
Abstract
Flow cytometry is the method of choice to 'diagnose' paroxysmal nocturnal hemoglobinuria (PNH) and has led to improved patient management. Most laboratories have limited experience with PNH testing, and many different flow approaches are used. Careful selection and validation of antibody conjugates has allowed the development of reagent cocktails suitable for detection of PNH RBCs, CD71+ reticulocytes, and WBCs in clinical/sub-clinical PNH samples. A CD235a-FITC/CD59-PE assay was developed capable of detecting Type III PNH RBCs at 0.01% ...
Source: Current Protocols in Cytometry - November 20, 2015 Category: Molecular Biology Tags: Curr Protoc Cytom Source Type: research
Uncompensated Polychromatic Analysis of Mitochondrial Membrane Potential Using JC-1 and Multilaser Excitation.
Authors: De Biasi S, Gibellini L, Cossarizza A
Abstract
The lipophilic cation JC-1 (5,5',6,6'-tetrachloro-1,1',3,3'-tetraethyl-benzimidazolyl carbocyanine iodide) has been used for more than 20 years as a specific dye for measuring mitochondrial membrane potential (ΔΨm ). In this unit, we revise our original protocol (that made use of a single 488 nm laser for the detection of monomers and aggregates, and where compensation was an important step) to use dual-laser excitation. Moreover, thanks to recently developed multilaser instruments and novel probes for surface and intracellular markers, JC-1 can be...
Source: Current Protocols in Cytometry - November 20, 2015 Category: Molecular Biology Tags: Curr Protoc Cytom Source Type: research
Cell Proliferation Method: Click Chemistry Based on BrdU Coupling for Multiplex Antibody Staining.
Authors: Cappella P, Gasparri F, Pulici M, Moll J
Abstract
Determination of incorporation of the thymidine analog 5-bromo-2'-deoxyuridine (BrdU) into DNA is a widely used method to analyze the cell cycle. However, DNA denaturation is required for BrdU detection with the consequence that most protein epitopes are destroyed and their immunocytochemical detection for multiplex analysis is not possible. A novel assay is presented for identifying cells in active S-phase that does not require the DNA denaturation step but nevertheless detects BrdU. For this purpose, cells were pulsed for a short time by 5-ethyny...
Source: Current Protocols in Cytometry - November 20, 2015 Category: Molecular Biology Tags: Curr Protoc Cytom Source Type: research
