Measurement of T-Cell Telomere Length Using Amplified-Signal FISH Staining and Flow Cytometry.
In this report, we describe a new approach for the measurement of telomere length within individual T cells using an amplified fluorescence in situ hybridization (FISH) technique (PrimeFlow RNA Assay). The unique aspect of this technique is that it amplifies the fluorescent signal rather than the target up to 3000-fold, resulting in the detection of as few as 1 copy of the target nucleic acid. While the current technique focuses on human T cells, this method can be broadly applied to a variety of cell types and disease models. © 2017 by John Wiley & Sons, Inc.
PMID: 28055115 [PubMed - in process] (Source: Curr...
Source: Current Protocols in Cytometry - January 6, 2017 Category: Molecular Biology Tags: Curr Protoc Cytom Source Type: research
Standardization, Calibration, and Control in Flow Cytometry.
Authors: Wang L, Hoffman RA
Abstract
Because flow cytometers are designed to measure particle characteristics, particles are the most common materials used to calibrate, control, and standardize the instruments. Definitions and cautions are provided for common terms to alert the reader to critical distinctions in meaning. This unit presents extensive background on particle types and cautions and describes practical aspects of methods to standardize and calibrate instruments. Procedures are provided to characterize performance in terms of optical alignment, fluorescence and light scatter resolution, and sen...
Source: Current Protocols in Cytometry - January 6, 2017 Category: Molecular Biology Tags: Curr Protoc Cytom Source Type: research
Automated Measurement of Blood Vessels in Tissues from Microscopy Images.
We describe a novel method of wall thickness calculation based on image skeletonization and compare its results to those of common techniques. Using both theoretical and empirical approaches, we demonstrate the accuracy and superiority of the skeleton-based method for measuring wall thickness while discussing its interpretation and limitations. Finally, we present a new freely available software tool, the VMI Calculator, to facilitate wall thickness measurements using our novel method. © 2016 by John Wiley & Sons, Inc.
PMID: 27723088 [PubMed - in process] (Source: Current Protocols in Cytometry)
Source: Current Protocols in Cytometry - October 12, 2016 Category: Molecular Biology Tags: Curr Protoc Cytom Source Type: research
Whole Blood Analysis of Leukocyte-Platelet Aggregates.
Authors: Gerrits AJ, Frelinger AL, Michelson AD
Abstract
In inflammatory and thrombotic syndromes, platelets aggregate with circulating leukocytes, especially monocytes and neutrophils. This leukocyte-platelet aggregate formation is initiated primarily through platelet surface expression of P-selectin (CD62P), following activation-dependent degranulation of α-granules, binding to its constitutively expressed counter-receptor, P-selectin glycoprotein ligand 1 (PSGL-1), on leukocytes. Monocyte-platelet aggregates are a more sensitive marker of platelet activation than platelet surface P-selectin. Detection ...
Source: Current Protocols in Cytometry - October 12, 2016 Category: Molecular Biology Tags: Curr Protoc Cytom Source Type: research
Flow Analysis and Sorting of Plant Chromosomes.
Authors: Vrána J, Cápal P, Šimková H, Karafiátová M, Čížková J, Doležel J
Abstract
Analysis and sorting of plant chromosomes (plant flow cytogenetics) is a special application of flow cytometry in plant genomics and its success depends critically on sample quality. This unit describes the methodology in a stepwise manner, starting with the induction of cell cycle synchrony and accumulation of dividing cells in mitotic metaphase, and continues with the preparation of suspensions of intact mitotic chromosomes, flow analysis and sorting of chromosomes, and finally processing of the sorted chromosom...
Source: Current Protocols in Cytometry - October 12, 2016 Category: Molecular Biology Tags: Curr Protoc Cytom Source Type: research
A Quantitative Method for Comparing the Brightness of Antibody-dye Reagents and Estimating Antibodies Bound per Cell.
We present a quantitative method for comparing the brightness of antibody-dye reagents and estimating antibodies bound per cell. The method is based on complementary binding of test and fill reagents to antibody capture microspheres. Several aliquots of antibody capture beads are stained with varying amounts of the test conjugate. The remaining binding sites on the beads are then filled with a second conjugate containing a different fluorophore. Finally, the fluorescence of the test conjugate compared to the fill conjugate is used to measure the relative brightness of the test conjugate. The fundamental assumption of the t...
Source: Current Protocols in Cytometry - July 2, 2016 Category: Molecular Biology Tags: Curr Protoc Cytom Source Type: research
High-Content Microscopy Analysis of Subcellular Structures: Assay Development and Application to Focal Adhesion Quantification.
Authors: Kroll T, Schmidt D, Schwanitz G, Ahmad M, Hamann J, Schlosser C, Lin YC, Böhm KJ, Tuckermann J, Ploubidou A
Abstract
High-content analysis (HCA) converts raw light microscopy images to quantitative data through the automated extraction, multiparametric analysis, and classification of the relevant information content. Combined with automated high-throughput image acquisition, HCA applied to the screening of chemicals or RNAi-reagents is termed high-content screening (HCS). Its power in quantifying cell phenotypes makes HCA applicable also to routine microscopy. However, developing effective HCA an...
Source: Current Protocols in Cytometry - July 2, 2016 Category: Molecular Biology Tags: Curr Protoc Cytom Source Type: research
Measurement and Characterization of Apoptosis by Flow Cytometry.
Authors: Telford W, Tamul K, Bradford J
Abstract
Apoptosis is an important mechanism in cell biology, playing a critical regulatory role in virtually every organ system. It has been particularly well characterized in the immune system, with roles ranging from immature immune cell development and selection to down-regulation of the mature immune response. Apoptosis is also the primary mechanism of action of anti-cancer drugs. Flow cytometry has been the method of choice for analyzing apoptosis in suspension cells for more than 25 years. Numerous assays have been devised to measure both the earliest and late...
Source: Current Protocols in Cytometry - July 2, 2016 Category: Molecular Biology Tags: Curr Protoc Cytom Source Type: research
Large Particle Sorting to Isolate Live Parasitic Nematode Eggs.
Authors: Schmidt A, Bouchery T, Le Gros G, Price KM
Abstract
Traditional jet-in-air cell sorters have been designed and optimized to isolate small particles such as mammalian lymphocytes with an average diameter of 10 μm. We discuss the practical considerations of setting up a conventional jet-in-air cell sorter, using a 200-μm nozzle, to isolate the large parasitic nematode eggs of Nippostrongylus brasiliensis, with a maximum size of 60 μm. The eggs were separated based on light scattering properties, no fluorescent dye or molecule was required. © 2016 by John Wiley & Sons, Inc.
PMID: 27...
Source: Current Protocols in Cytometry - April 3, 2016 Category: Molecular Biology Tags: Curr Protoc Cytom Source Type: research
Measurement of Low-Abundance Intracellular mRNA Using Amplified FISH Staining and Image-Based Flow Cytometry.
Authors: Henning AL, Sampson JN, McFarlin BK
Abstract
Recent advances in instrument design and reagent development have enabled the rapid progression in available measurement techniques in the field of flow cytometry. In particular, image-based flow cytometry extends the analysis capacity found in traditional flow cytometry. Until recently, it was not possible to measure intracellular mRNA in specific phenotypes of cells by flow cytometry. In this protocol, a method of completing simultaneous intracellular measurement of mRNA and protein for PPAR-gamma in peripheral blood monocytes, which have been exposed...
Source: Current Protocols in Cytometry - April 3, 2016 Category: Molecular Biology Tags: Curr Protoc Cytom Source Type: research
Quantitative Flow Cytometry Measurements in Antibodies Bound per Cell Based on a CD4 Reference.
Authors: Wang L, Degheidy H, Abbasi F, Mostowski H, Marti G, Bauer S, Hoffman RA, Gaigalas AK
Abstract
Multicolor flow cytometer assays with fluorescently labeled antibodies are routinely used in clinical laboratories to measure the cell number of specific immunophenotypes and to estimate expression levels of specific receptors/antigens either on the cell surface or intracellularly. The cell number and specific receptors/antigens serve as biomarkers for pathological conditions at various stages of a disease. Existing methods and cell reference materials for quantitative expression measurements have not yet...
Source: Current Protocols in Cytometry - January 16, 2016 Category: Molecular Biology Tags: Curr Protoc Cytom Source Type: research
Optimized MOL-PCR for Characterization of Microbial Pathogens.
Authors: Wuyts V, Roosens NH, Bertrand S, Marchal K, De Keersmaecker SC
Abstract
Characterization of microbial pathogens is necessary for surveillance, outbreak detection, and tracing of outbreak sources. This unit describes a multiplex oligonucleotide ligation-PCR (MOL-PCR) optimized for characterization of microbial pathogens. With MOL-PCR, different types of markers, like unique sequences, single-nucleotide polymorphisms (SNPs) and indels, can be simultaneously analyzed in one assay. This assay consists of a multiplex ligation for detection of the markers, a singleplex PCR for signal amplification, and ...
Source: Current Protocols in Cytometry - January 16, 2016 Category: Molecular Biology Tags: Curr Protoc Cytom Source Type: research
Simultaneous, Single-Cell Measurement of Messenger RNA, Cell Surface Proteins, and Intracellular Proteins.
Authors: Soh KT, Tario JD, Colligan S, Maguire O, Pan D, Minderman H, Wallace PK
Abstract
Nucleic acid content can be quantified by flow cytometry through the use of intercalating compounds; however, measuring the presence of specific sequences has hitherto been difficult to achieve by this methodology. The primary obstacle to detecting discrete nucleic acid sequences by flow cytometry is their low quantity and the presence of high background signals, rendering the detection of hybridized fluorescent probes challenging. Amplification of nucleic acid sequences by molecular techniques such as in situ PCR hav...
Source: Current Protocols in Cytometry - January 16, 2016 Category: Molecular Biology Tags: Curr Protoc Cytom Source Type: research
Overview of very small embryonic-like stem cells (VSELs) and methodology of their identification and isolation by flow cytometric methods.
We describe the recommended steps in detail for their successful identification and isolation from adult tissues. These protocols were initially established to isolate such cells from murine bone marrow (BM) and human cord blood (CB) and may also be employed to isolate these primitive cells from other adult organs and embryonic tissues. Here, we focus on some critical parameters/key points required for the successful identification and purification of these rare cells by employing classical flow cytometry. In the last part of this unit, we also discuss a novel flow cytometric tool, ImageStream, an imaging flow cytometer, w...
Source: Current Protocols in Cytometry - November 20, 2015 Category: Molecular Biology Tags: Curr Protoc Cytom Source Type: research
Stem cell side population analysis and sorting using DyeCycle violet.
Authors: Telford WG
Abstract
Hoechst side population (SP) analysis has proven to be a valuable technique for identifying and sorting stem and early progenitor cells in a variety of tissues and species. In this method, the DNA binding dye Hoechst 33342 is loaded into the cell population of interest; stem cells preferentially exclude this dye, and these low-fluorescence cells can be detected by flow cytometry. However, Hoechst SP analysis usually requires a flow cytometer equipped with an ultraviolet laser source for optimal performance. Unfortunately, ultraviolet lasers are expensive and are not common fixt...
Source: Current Protocols in Cytometry - November 20, 2015 Category: Molecular Biology Tags: Curr Protoc Cytom Source Type: research
