Current Protocols in Cytometry最新文献

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 cytometers. Here, we present optimized protocols for EV labeling that increase the signal-to-noise ratio of EVs by removing residual antibody. Protocols for alignment of high-resolution jet-in-air flow cytometers are also provided. Published 2020. U.S. Government.

Basic Protocol 1: Bulk EV staining with CFSE protein binding dye

Basic Protocol 2: Antigen-specific staining of EV markers with fluorochrome-conjugated antibodies

Basic Protocol 3: Astrios EQ instrument setup and sample acquisition

Basic Protocol 4: Counting particles and EVs on Astrios EQ with spike-in reference beads

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.

Basic Protocol 1: Live imaging of lung slices

Support Protocol 1: Staining lung sections with fluorescent antibodies

Basic Protocol 2: Live imaging in the mouse lung

Support Protocol 2: Intratracheal instillations

Support Protocol 3: Intravascular instillations

Support Protocol 4: Monitoring vital signs of the mouse during live lung imaging

Support Protocol 5: Antibodies

Support Protocol 6: Fluorescent reporter mice

Basic Protocol 3: Quantification of neutrophil-platelet aggregation in pulmonary vasculature

Basic Protocol 4: Quantification of platelet-dependent pulmonary thrombosis

Basic Protocol 5: Quantification of pulmonary vascular permeability

Use of flow cytometry to analyze small particles has been implemented for several decades. More recently, small particle analysis has become increasingly utilized owing to the increased sensitivity of conventional and commercially available flow cytometers along with growing interest in small particles such as extracellular vesicles (EVs). Despite an increase in small particle flow cytometry utilization, a lack of standardization in data reporting has resulted in a growing body of literature regarding EVs that cannot be easily interpreted, validated, or reproduced. Methods for fluorescence and light scatter standardization are well established, and the reagents to perform these analyses are commercially available. Here, we describe FCM_PASS , a software package for performing fluorescence and light scatter calibration of small particles while generating standard reports conforming to the MIFlowCyt-EV standard reporting framework. This article covers the workflow of implementing calibration using FCM_PASS 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 gating of fluorescence calibration materials Alternate Protocol: Cross-calibration of fluorescence reference materials Basic Protocol 3: Cataloguing light scatter calibration materials Basic Protocol 4: Cataloguing fluorescence calibration materials Basic Protocol 5: Creating cytometer databases and datasets Basic Protocol 6: Importing fcs files Basic Protocol 7: Fluorescence calibration Basic Protocol 8: Light scatter calibration Basic Protocol 9: Performing and reporting fcs file calibration.

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 highlight patterns of normal maturation and allow identification of neoplastic hematopoietic progenitor populations with a high degree of sensitivity and specificity. The method was developed in a clinical laboratory setting for the diagnosis of myeloid stem cell disorders and neoplasms, and has been extensively validated both technically and clinically for the detection of MRD in AML. © 2020 The Authors. Basic Protocol: Staining and flow cytometry for AML minimal residual disease detection Support Protocol: Analysis and interpretation of data for AML minimal residual disease detection.

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-intensity classification. Here, we describe a novel strategy for flow cytometric quantification of GAP activity in which staining and analytical flow cytometry facilitate the detection and quantification of subpopulations of leukocytes with different GAP activities. Our experiments demonstrate the potential of flow cytometry as a simple and highly sensitive approach for measuring GAP activity in unlysed whole blood. Notably, a comparison of flow cytometry and enzyme cytochemistry techniques showed that enzyme activity scores were not similar, indicating that results needs to be interpreted with caution, given that the enzyme-substrate binding affinities may differ, as well as the subjective evaluation of the intensity of the precipitated dye. © 2020 Wiley Periodicals LLC. Basic Protocol: Protocol preparation, sample acquisition, and gating strategy for flow cytometric identification of alkaline phosphatase activity in granulocytes from whole blood samples Support Protocol 1: Sample preparation for granulocyte alkaline phosphatase determination by flow cytometry using no-lyse no-wash methods Support Protocol 2: Data analysis and formula to calculate the GAP score.

For microorganisms in particular, viability is a term that is difficult to define and a state consequently difficult to measure. The traditional (and gold standard) usage equates viability and culturability (i.e., the ability to multiply) but the process of determining culturability is often too slow. Flow cytometry provides the opportunity to make rapid and quantitative measurements of dye uptake in large numbers of cells and we can therefore exploit the flow cytometric approach to evaluate so-called viability stains and to develop protocols for more routine assessments of microbial viability. 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.

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 methodology to prepare samples for polychromatic flow cytometry. The use of multiple washing steps and rigorous controls creates high-quality data with good separation between cell populations. Experimental data acquired using this protocol can be analyzed via computational algorithms that perform end-to-end analysis. © 2020 by John Wiley & Sons, Inc. Basic Protocol 1: Preparation of single-cell suspension for flow cytometry Support Protocol 1: Lung preparation Support Protocol 2: Counting cells on a flow cytometer Basic Protocol 2: Surface and intracellular flow cytometry staining Support Protocol 3: Single-color bead controls.