Imaging Flow Cytometry: Methods And Protocols WORK
Measurement of γ-H2AX foci levels in cells provides a sensitive and reliable method for quantitation of the radiation-induced DNA damage response. The objective of the present study was to develop a rapid, high-throughput γ-H2AX assay based on imaging flow cytometry (IFC) using the ImageStreamX Mk II (ISX) platform to evaluate DNA double strand break (DSB) repair kinetics in human peripheral blood cells after exposure to ionizing irradiation.
Imaging Flow Cytometry: Methods and Protocols
Overall, further development and validation of the IFC-based γ-H2AX assay system presented in this work will allow for evaluation of DNA damage and DSB repair capacity with increased resolution, sensitivity, accuracy and high-speed image acquisition as compared to traditional flow cytometry and traditional microscope immunohistochemical methods [28, 30]. End-to-end automation of the IFC-based γ-H2AX assay can be achieved with the integration of our RABiT (Rapid Automated Biodosimetry Technology) platform for automated sample preparation from small volumes of blood . Measurements of individual DSB repair capacity within a large population could offer valuable information to advance this high-throughput assay for translational research such as monitoring risk and response among radiotherapy patients.
The study of extracellular vesicles (EVs) has evolved into a compelling field of intercellular communication and pathophysiology research. The submicron size of EVs and the complexity of the biofluids in which they are suspended confound their analysis by traditional light microscopy or flow cytometry. Observation and quantitation of EVs have been achieved with Amnis imaging flow cytometers (IFCs) with reduced volumes and sample preparation times, suggesting an auspicious role for IFCs in the advancement of EV biology.
Gating with non-imaging flow cytometers relies on the degree of light scatter and fluorescence intensity, which leads to the frequent, inadvertent inclusion of contaminating particles in the presumed EV population. IFCs exploit the ability to connect the quantitative morphological characteristics of an object to its image in the image gallery to confirm and refine gating. This capability has already led to the confirmation of some existing EV gating strategies, while challenging others.
EVs are now believed to be significant mediators in the pathogenesis of neurological, vascular, hematological and autoimmune disease. Moreover, EVs are considered key to tumorigenesis, metastasis, immunomodulation and other cancer processes that may contribute to poor disease outcomes. In order for the assessment of EVs to transition into the clinic to aid in diagnosis and treatment, it is essential to identify technologies that will facilitate the development of standard assays for quantitation and characterization. Although sensitive enough to resolve tiny cellular particles, high-magnification microscopy is limited by the typical dispersal of microvesicles in biofluids, as well as by a lack of quantitative data. Non-imaging flow cytometers are well suited for measuring objects in suspension but are generally unable to detect particles less than 400 nm in diameter, and they cannot provide visual confirmation of particle integrity for confident characterization.
In this case study, researchers at the Weizmann Institute of Science in Israel developed and optimized an imaging flow cytometry (IFC) protocol to provide a high-throughput, quantitative and highly robust method for studying viral infection and virus-host interactions.
In order to explore the temporal progression of events in infected cells, Minsky and team used imaging flow cytometry (IFC) to monitor the changes in Acanthamoeba polyphaga (amoeba) cells that had been infected with the giant Mimivirus.
High-throughput single cell imaging is a critical enabling and driving technology in molecular and cellular biology, biotechnology, medicine and related areas. Imaging flow cytometry combines the single-cell imaging capabilities of microscopy with the high-throughput capabilities of conventional flow cytometry. Recent advances in imaging flow cytometry are remarkably revolutionizing single-cell analysis. This article describes recent imaging flow cytometry technologies and their challenges.
Spectre is an R package and computational toolkit that enables comprehensive end-to-end integration, exploration, and analysis of high-dimensional cytometry, spatial/imaging, or single-cell data from different batches or experiments. Spectre streamlines the analytical stages of raw data pre-processing, batch alignment/integration, clustering, dimensionality reduction, visualisation, population annotation, and quantitative/statistical analysis; with a simple, clear, and modular design of analysis workflows, that can be utilised by both data and laboratory scientists.
The use of the annexin V apoptosis assay protocol is a common method for detecting apoptotic cells. The below protocols are recommended for use with the specific flow cytometry kits mentioned. Please see the Annexin V Staining page for a discussion about general experimental conditions and avoiding false positives, or to review a selection guide for all of our annexin V products.
Unlike assays using BrdU staining, Click-iT EdU assays are not antibody-based and therefore do not require DNA denaturation for detection of the incorporated nucleoside. In addition, for increased utility, the Click-iT Plus EdU assay can be multiplexed with R-phycoerythrin (R-PE) and R-PE tandems, fluorescent proteins (GFP and mCherry), and also with BrdU in a BrdU and EdU double staining experiment. The Click-iT EdU technology has not only been developed into kits for flow cytometry and imaging applications (including HCS), but it also has been adapted for the colorimetric detection of EdU in an immunohistochemistry (IHC) assay. When you compare the methods side-by-side (Figures 1 and 2), the benefits of the Click-iT EdU assay and Invitrogen Click-iT Plus EdU assay for cell proliferation are clear.
Figure 2. EdU staining with Click-iT and Click-iT Plus detection. EdU that is incorporated into newly synthesized DNA is detected without the need for DNA denaturation. Simple, easy-to-follow, robust protocols allow for reproducible, bright staining. The Click-iT EdU Assay kits work with most standard fluorophore conjugates. Click-iT Plus EdU Assay kits were designed for maximum multiplex flexibility and are compatible with R-PE (and tandems) and fluorescent proteins such as GFP and mCherry. Protocol shown is for processing rat tissue sections. Optimized kits developed for use in flow cytometry, imaging, microplates, high-content screening, and colorimetric immunohistochemistry applications.
The copper concentrations typically used in traditional click chemistry reactions can affect fluorophores such as GFP, mCherry, R-PE, and R-PE tandem dyes. The Click-iT Plus formulation may be effectively employed in a low-copper reaction, enabling increased multiplexability compared to the original Click-iT EdU assays. Click-iT Plus EdU assays can be used in conjunction with R-PE and R-PE tandems, as well as fluorescent proteins such as GFP and mCherry, without loss of the accuracy or speed of the original Click-iT EdU assay. The low-copper reaction with Click-iT Plus EdU results in bright signals and retains the fluorescent signal from GFP (Figure 5). In addition, detection of EdU with the Click-iT Plus Alexa Fluor 488 picolyl azide was compatible with PE-Cy7 fluorescence in a flow cytometric assay for cell proliferation (Figure 6). Select from Click-iT Plus EdU kits for flow cytometry or Click-iT Plus EdU assays for imaging applications.
The continuous improvement of imaging technologies has driven the development of sophisticated reporters to monitor biological processes. Such constructs should ideally be assembled in a flexible enough way to allow for their optimization. Here we describe a highly reliable cloning method to efficiently assemble constructs for imaging or flow cytometry applications in mammalian cell culture systems. We bioinformatically identified a list of restriction enzymes whose sites are rarely found in human and mouse cDNA libraries. From the best candidates, we chose an enzyme combination (MluI, XhoI and SalI: MXS) that enables iterative chaining of individual building blocks. The ligation scar resulting from the compatible XhoI- and SalI-sticky ends can be translated and hence enables easy in-frame cloning of coding sequences. The robustness of the MXS-chaining approach was validated by assembling constructs up to 20 kb long and comprising up to 34 individual building blocks. By assessing the success rate of 400 ligation reactions, we determined cloning efficiency to be 90% on average. Large polycistronic constructs for single-cell imaging or flow cytometry applications were generated to demonstrate the versatility of the MXS-chaining approach. We devised several constructs that fluorescently label subcellular structures, an adapted version of FUCCI (fluorescent, ubiquitination-based cell cycle indicator) optimized to visualize cell cycle progression in mouse embryonic stem cells and an array of artificial promoters enabling dosage of doxycyline-inducible transgene expression. We made publicly available through the Addgene repository a comprehensive set of MXS-building blocks comprising custom vectors, a set of fluorescent proteins, constitutive promoters, polyadenylation signals, selection cassettes and tools for inducible gene expression. Finally, detailed guidelines describe how to chain together prebuilt MXS-building blocks and how to generate new customized MXS-building blocks.
Here we describe a chaining method that is specifically designed for the assembly of constructs for imaging and flow cytometry approaches in mammalian cell culture systems. We offer a comprehensive library of parts and cassettes through the Addgene repository ( ) to allow researchers to specifically tailor constructs to their needs. To demonstrate the versatility of the technique, we engineered several constructs that fluorescently label cellular landmarks, an adaptation of the fluorescent, ubiquitination-based cell cycle indicator (FUCCI)  to mouse embryonic stem cells and an array of artificial doxycyline-inducible promoters . 041b061a72