Fluorescent DNA-binding Dye Research Articles

Tachyplesin Causes Membrane Instability That Kills Multidrug-Resistant Bacteria by Inhibiting the 3-Ketoacyl Carrier Protein Reductase FabG.

Tachyplesin is a type of cationic β-hairpin antimicrobial peptide discovered in horseshoe crab approximately 30 years ago that is well known for both its potential antimicrobial activities against multidrug-resistant bacteria and its cytotoxicity to mammalian cells. Though its physical interactions with artificial membranes have been well studied, details of its physiological mechanism of action the physiological consequences of its action remain limited. By using the DNA-binding fluorescent dye propidium iodide to monitor membrane integrity, confocal microscopy to assess the intracellular location of FITC-tagged tachyplesin, and RNA sequencing of the differentially expressed genes in four Gram-negative bacteria (Escherichia coli, Acinetobacter baumannii, Klebsiella pneumoniae, and Pseudomonas aeruginosa) treated with lethal or sublethal concentrations of tachyplesin, we found that compared with levofloxacin-treated bacteria, tachyplesin-treated bacteria showed significant effects on the pathways underlying unsaturated fatty acid biosynthesis. Notably, RNA levels of the conserved and essential 3-ketoacyl carrier protein reductase in this pathway (gene FabG) were elevated in all of the four bacteria after tachyplesin treatment. In vitro tests including surface plasmon resonance and enzyme activity assays showed that tachyplesin could bind and inhibit 3-ketoacyl carrier protein reductase, which was consistent with molecular docking prediction results. As unsaturated fatty acids are important for membrane fluidity, our results provided one possible mechanism to explain how tachyplesin kills bacteria and causes cytotoxicity by targeting membranes, which may be helpful for designing more specific and safer antibiotics based on the function of tachyplesin.

Biomarkers in Cerebrospinal Fluid: Analysis of Cell-Free Circulating Mitochondrial DNA by Digital PCR.

Cerebrospinal fluid (CSF) contains molecules directly linked with brain function because it permeates brain tissue. The analysis of protein biomarkers in CSF is currently recommended for the diagnosis of neurodegenerative disorders, but the clinical sensitivity and specificity are still being investigated. A major drawback is that most of the currently used biomarkers of neurodegenerative diseases are proteins that are found at very low concentrations in CSF and need to be measured by immunoassays that provide relative values, which sometimes are difficult to reproduce between laboratories. In contrast, the recent availability of digital PCR platforms allows the absolute quantification of nucleic acids at single-molecule resolution, but their presence in CSF has not been characterized. CSF contains cell-free mitochondrial DNA (mtDNA) and changes in the concentration of this nucleic acid are linked to neurodegeneration. Here we describe a method to measure the concentration of cell-free circulating mtDNA directly in unpurified CSF using droplet digital PCR with either hydrolysis probes or fluorescent DNA-binding dye methods. This protocol allows the detection and absolute quantification of mtDNA content in the CSF with high analytical sensitivity, specificity, and accuracy.

Isolation of Cancer Stem Cells by Side Population Method.

The Hoechst side population (SP) method is a flow cytometry technique used to obtain stem cells based on the dye efflux properties of the ATP-binding cassette (ABC) transporters. The SP cells are characterized by their capability to efflux the fluorescent DNA-binding dye Hoechst 33342 through their ABC transporters and are enriched in stem cells, which are endowed with a self-renewal capacity and multilineage differentiation potential and express the stemness genes including ABC multidrug transporters. The protocols outlined in this book chapter describe the isolation method of the SP cells from human lung carcinoma cell lines by using Hoechst 33342. In addition, we refer to the propagation method of SP cells by successive rounds of fluorescence-activated cell sorting analysis for SP cells. These approaches will be helpful for the establishment of novel in vitro and in vivo models using cancer stem cells, which may play a key role during carcinogenesis and/or tumor progression.

A Standardized Approach for Multispecies Purification of Mammalian Male Germ Cells by Mechanical Tissue Dissociation and Flow Cytometry

Fluorescence-activated cell sorting (FACS) has been one of the methods of choice to isolate enriched populations of mammalian testicular germ cells. Currently, it allows the discrimination of up to 9 murine germ cell populations with high yield and purity. This high-resolution in discrimination and purification is possible due to unique changes in chromatin structure and quantity throughout spermatogenesis. These patterns can be captured by flow cytometry of male germ cells stained with fluorescent DNA-binding dyes such as Hoechst-33342 (Hoechst). Herein is a detailed description of a recently developed protocol to isolate mammalian testicular germ cells. Briefly, single cell suspensions are generated from testicular tissue by mechanical dissociation, double stained with Hoechst and propidium iodide (PI) and processed by flow cytometry. A serial gating strategy, including the selection of live cells (PI negative) with different DNA content (Hoechst intensity), is used during FACS sorting to discriminate up to 5 germ cell types. These include, with corresponding average purities (determined by microscopy evaluation): spermatogonia (66%), primary (71%) and secondary (85%) spermatocytes, and spermatids (90%), further separated into round (93%) and elongating (87%) subpopulations. Execution of the entire workflow is straightforward, allows the isolation of 4 cell types simultaneously with the appropriate FACS machine, and can be performed in less than 2 h. As reduced processing time is crucial to preserve the physiology of ex vivo cells, this method is ideal for downstream high-throughput studies of male germ cell biology. Moreover, a standardized protocol for multispecies purification of mammalian germ cells eliminates methodological sources of variables and allows a single set of reagents to be used for different animal models.

Identification and gene expression analysis of the side population subclone in mantle cell lymphoma

Introduction: It has been increasingly understood that seemingly effective treatments merely remove the bulk of tumor cells, while more rare cancer stem cells reside. The treatment resistant cells soon repopulate and form new tumor mass. These are often selected by the exposure to therapy and thus increasingly resistant. One way of identifying such cells is through analysis of the side population (SP) in tumor masses. In this study, we investigated the presence of SP in twenty-five lymphoma cell lines, representing five different types of B-cell lymphomas. Methods: Cells were labelled with fluorescent DNA-binding dye Hoechst 33342, and SP cells were identified as a subpopulation of cells that have an elevated rate of Hoechst efflux. All samples were analyzed on a FACS Aria II (BD Bioscience, San Jose, CA, USA). Using cell sorting, both SP and non-SP cells could be isolated. The presence of SP was assessed in MCL patient samples and cell lines derived from MCL, FL, DLBCL, Burkitt's lymphoma (BL), and CLL. Subsequent gene expression analysis was performed using Affymetrix Human Transcriptome Array 2.0 (HTA 2.0; Affymetrix Inc, Santa Clara, CA, USA). Results: The presence of SP was limited to one mantle cell lymphoma (MCL) derived primary sample (P3) and cell line (REC-1). Downstream analysis involving in vitro cultivation of sorted SP shows that SP cells are able to grow and differentiate into daughter cells and non-SP cells. Of interest, the frequency of SP cells was enriched when wild type REC-1 cells were exposed and became resistant to chemotherapy. Moreover, a differential gene expression profile between SP cells and non-SP cells was revealed by comprehensive gene expression analysis showing deregulation of group of genes involved in cell proliferation, cell cycle regulation, cell migration, BCR signaling, cancer progression, and tumorigenesis. Of major importance, validation of gene expression results reveal that SP, compared to non-SP, exhibits significantly higher expression levels of CD44 and CD69, known to be involved in cancer stem cells and lymphocyte proliferation, respectively. Conclusion: We show that SP is found among MCL patients and that genes involved in several cell signaling pathways are deregulated in SP derived from both REC-1 and patient material. This indicates that REC-1 can serve as a tool for future screening efforts. Importantly, we show evidence that CD69 and CD44 are commonly upregulated in SP cells from MCL patient material and could have important functional roles in relapse and treatment resistance. Keywords: flow cytometry; mantle cell lymphoma (MCL)

MicroRNA-192-5p Promote the Proliferation and Metastasis of Hepatocellular Carcinoma Cell by Targeting SEMA3A.

Side population (SP) cells are a small subset of cells isolated from a cultured cancer cell line with characteristics similar to those of cancer stem cells, such as high metastatic and tumorigenic potentials. However, the molecular mechanisms remain unclear for the malignant properties of SP cells. In this study, SP cells were isolated by staining cultured HCCLM3 cells with fluorescent DNA-binding dye Hoechst 33342 and sorted by flow cytometry. The proportion of SP cells was 2.79%±0.19% in the HCCLM3 cell line. Compared with non-SP cells, SP cells possessed stronger capability of sphere formation and tumorigenicity, and expressed higher levels of CD133 and CD90. Then, we found that SP cells possessed 25 upregulated and 34 downregulated microRNAs with differences of >3-fold. As one of the upregulated microRNAs, miR-192-5p was computationally predicted to target semaphorin 3A (SEMA3A), a potent suppressor of tumor angiogenesis in various cancer models. Luciferase reporter assay showed that SEMA3A was a direct target of miR-192-5p. Overexpression of miR-192-5p promoted cell proliferation and metastasis targeting SEMA3A in HCCLM3 cells. Immunohistochemical staining revealed that SEMA3A expression was significantly reverse associated with metastasis in hepatocellular carcinoma tissues. The results indicate that miR-192-5p contributes to targeting SEMA3A in HCCLM3 cells, and this may be used as a target in targeted therapy and a marker for cancer behavior and prognosis.

Rapid Molecular Diagnosis of Bacterial Infection Using Integrated Lab-on-a-disc

Bacterial infection is a disaster leading to high fatal rate in intensive caring unit. Rapid profiling of infectious bacteria is necessary for applying the correct medication. Current gold standard using plate inoculation is inaccurate, time-consuming and labour-intensive. Therefore we have developed a molecular diagnostic approach to target marker DNA of the infectious bacteria for rapid profiling. A micro-fluidic platform lab-on-a-disc (LOAD) has been adopted because using one simple spinning action can provide highly controllable centrifugation drive force for the actuation of samples and reagents anywhere within the boundary of the disc. When centrifugal force-triggered valve is applied, complex sequential flow of liquid can be controlled with various centrifugal force. This will enable parallel execution of many reactions simultaneously with minimal complexity in the design of fluidic pumping and flow control.We report an integrated LOAD for direct sample-to-answer applications - fully automated assay from patient's sample input to detection of signal output. The integrated LOAD with PDMS-made microfludic disc performs three major functions, namely DNA extraction, LAMP reaction and detection. Using microfluidics technology, target bacteria can be detected using as little as 10 μL blood sample loaded into sample loading site. The DNA release after cell lysis in heating site is bound on the silica membrane. After washing, the purified DNA elution is subjected to LAMP reaction to amplify the target genetic sequence. Loop-mediated isothermal amplification (LAMP) is an isothermal nucleic acid amplification method where reaction occurs under 65 oC. The amplified signal is reported by DNA binding fluorescent dye. Our prototype shows high yield and purity of bacterial DNA from clinical samples such as blood. We demonstrated the detection of Acinetobacter baumanii, which is one of the key pathogens resulting in hospital-acquired infections, in clinical blood sample using the LOAD platform. Fast signal detection and active temperature control within the LOAD platform has also enabled real-time LAMP targeting of specific DNA sequences as barcodes to identify infected bacterial species. We found the detection sensitivity of LAMP using DNA is 10-15 g, while that of bacteria concentration is 102 cfu/ml. The system is capable of providing bacterial DNA profiling within 2 hours.In conclusion, our integrated LOAD is a simple (sample-to-answer), specific (specific genetic sequences recognition), robust (automated assay on microfluidic disc) method for rapid molecular diagnosis of bacterial infection. The short turnaround time and the technical advancement of sample-to-answer in one LOAD platform approach for rapid bacterial detection should have much potential in addressing the needs of point-of-care medical diagnosis applications. The simplicity allows the clinical healthcare workers to utilize without needing technical training.

Antifungal Activity of Oleuropein against Candida albicans-The In Vitro Study.

In the present study we investigated activity of oleuropein, a complex phenol present in large quantities in olive tree products, against opportunistic fungal pathogen Candida albicans. Oleuropein was found to have in vitro antifungal activity with a minimal inhibitory concentration (MIC) value of 12.5 mg·mL−1. Morphological changes in the nuclei after staining with fluorescent DNA-binding dyes revealed that apoptosis was a primary mode of cell death in the analyzed samples treated with subinhibitory concentrations of oleuropein. Our results suggest that this antifungal agent targets virulence factors essential for establishment of the fungal infection. We noticed that oleuropein modulates morphogenetic conversion and inhibits filamentation of C. albicans. The hydrophobicity assay showed that oleuropein in sub-MIC values has significantly decreased, in both aerobic and anaerobic conditions, the cellular surface hydrophobicity (CSH) of C. albicans, a factor associated with adhesion to epithelial cells. It was also demonstrated that the tested compound inhibits the activity of SAPs, cellular enzymes secreted by C. albicans, which are reported to be related to the pathogenicity of the fungi. Additionally, we detected that oleuropein causes a reduction in total sterol content in the membrane of C. albicans cells, which might be involved in the mechanism of its antifungal activity.

Circulating MicroRNA Quantification Using DNA-binding Dye Chemistry and Droplet Digital PCR

Circulating (of cell-free) microRNAs (miRNAs) are released from cells into the blood stream. The amount of specific microRNAs in the circulation has been linked to a disease state and has the potential to be used as disease biomarker. A sensitive and accurate method for circulating microRNA quantification using a dye-based chemistry and droplet digital PCR technology has been recently developed. Specifically, using Locked Nucleic Acid (LNA)-based miRNA-specific primers with a green fluorescent DNA-binding dye in a compatible droplet digital PCR system it is possible to obtain the absolute quantification of specific miRNAs. Here, we describe how performing this technique to assess miRNA amount in biological fluids, such as plasma and serum, is both feasible and effective.

Is biofilm removal properly assessed? Comparison of different quantification methods in a 96-well plate system.

Various methods have been reported to quantify total biofilm or different components of biofilm; however, these methods are often confusedly used, leading to discrepancies and misleading results. In this study, different methods for quantification of biofilm, including those for total biomass, total amount of bacterial cells, viable cell number, and amount of extracellular polymeric substances, were systematically compared in microtiter plates. To evaluate which method is suitable for assessment of biofilm removal and for bacterial killing, biofilm samples were treated with various cleaners possessing removing and/or killing capacities. It was found that most of the methods tested in this study in general exhibited high reproducibility and repeatability. Crystal Violet staining was a simple but reliable method for total biomass quantification. Total bacteria cell numbers could be reliably quantified by the fluorescent DNA-binding dye Acridine Orange. Viable cells could be quantified by either an ATP-based assay or a proliferation assay. Both of these viability methods showed a broad detection range and led to precise measurement. For quantification of proteins in the biofilm, staining with fluorescein isothiocyanate was most suitable. Furthermore, it was revealed that a combination of different methods is required to determine if a cleaner kills or removes biofilm.Electronic supplementary materialThe online version of this article (doi:10.1007/s00253-016-7396-9) contains supplementary material, which is available to authorized users.

High-Resolution Melt Curve Analysis in Cancer Mutation Screen.

High-resolution melt (HRM) curve analysis is a PCR-based assay that identifies sequence alterations based on subtle variations in the melting curves of mutated versus wild-type DNA sequences. HRM analysis is a high-throughput, sensitive, and efficient alternative to Sanger sequencing and is used to assess for mutations in clinically important genes involved in cancer diagnosis. The technique involves PCR amplification of a target sequence in the presence of a fluorescent double-stranded DNA (dsDNA) binding dye, melting of the fluorescent amplicons, and subsequent interpretation of melt curve profiles.

The Analysis of Cell Cycle, Proliferation, and Asymmetric Cell Division by Imaging Flow Cytometry.

Measuring cellular DNA content by conventional flow cytometry (CFC) and fluorescent DNA-binding dyes is a highly robust method for analysing cell cycle distributions within heterogeneous populations. However, any conclusions drawn from single-parameter DNA analysis alone can often be confounded by the asynchronous nature of cell proliferation. We have shown that by combining fluorescent DNA stains with proliferation tracking dyes and antigenic staining for mitotic cells one can elucidate the division history and cell cycle position of any cell within an asynchronously dividing population. Furthermore if one applies this panel to an imaging flow cytometry (IFC) system then the spatial information allows resolution of the four main mitotic phases and the ability to study molecular distributions within these populations. We have employed such an approach to study the prevalence of asymmetric cell division (ACD) within activated immune cells by measuring the distribution of key fate determining molecules across the plane of cytokinesis in a high-throughput, objective, and internally controlled manner. Moreover the ability to perform high-resolution, temporal dissection of the cell division process lends itself perfectly to investigating the influence chemotherapeutic agents exert on the proliferative capacity of transformed cell lines. Here we describe the method in detail and its application to both ACD and general cell cycle analysis.

An Imaging Flow Cytometry-based approach to analyse the fission yeast cell cycle in fixed cells

Fission yeast (Schizosaccharomyces pombe) is an excellent model organism for studying eukaryotic cell division because many of the underlying principles and key regulators of cell cycle biology are conserved from yeast to humans. As such it can be employed as tool for understanding complex human diseases that arise from dis-regulation in cell cycle controls, including cancers. Conventional Flow Cytometry (CFC) is a high-throughput, multi-parameter, fluorescence-based single cell analysis technology. It is widely used for studying the mammalian cell cycle both in the context of the normal and disease states by measuring changes in DNA content during the transition through G1, S and G2/M using fluorescent DNA-binding dyes. Unfortunately analysis of the fission yeast cell cycle by CFC is not straightforward because, unlike mammalian cells, cytokinesis occurs after S-phase meaning that bi-nucleated G1 cells have the same DNA content as mono-nucleated G2 cells and cannot be distinguished using total integrated fluorescence (pulse area). It has been elegantly shown that the width of the DNA pulse can be used to distinguish G2 cells with a single 2C foci versus G1 cells with two 1C foci, however the accuracy of this measurement is dependent on the orientation of the cell as it traverses the laser beam. To this end we sought to improve the accuracy of the fission yeast cell cycle analysis and have developed an Imaging Flow Cytometry (IFC)-based method that is able to preserve the high throughput, objective analysis afforded by CFC in combination with the spatial and morphometric information provide by microscopy. We have been able to derive an analysis framework for subdividing the yeast cell cycle that is based on intensiometric and morphometric measurements and is thus robust against orientation-based miss-classification. In addition we can employ image-based metrics to define populations of septated/bi-nucleated cells and measure cellular dimensions. To our knowledge, this is the first use of IFC to study fission yeast and we are confident that this will provide a springboard for further IFC-based analysis across all aspects of fission yeast biology.

Agglutination Assays of the Plasmodium falciparum-Infected Erythrocyte.

The agglutination assay is used to determine the ability of antibodies to recognize parasite variant antigens on the surface of Plasmodium falciparum-infected erythrocytes. In this technique, infected erythrocytes are selectively labelled with a DNA-binding fluorescent dye and mixed with antibodies of interest to allow antibody-surface antigen binding. Recognition of surface antigens by the antibodies can result in the formation of agglutinates containing multiple parasite-infected erythrocytes. These can be viewed and quantified using a fluorescence microscope.

Tibetan PHD2D4E High Altitude Adapted Gene Can be Rapidly Detected By High Resolution Melting Assay

High altitude is accompanied by hypoxia. Acute and chronic hypoxia induces a number of compensatory physiological responses mediated by hypoxia-inducible factors (HIFs) that regulate erythropoiesis, iron and energy metabolism, and other essential organismal responses. Excessive HIF responses occurring at high altitude may be accompanied by morbidity (polycythemia and pulmonary hypertension) or mortality (brain and pulmonary edema). HIFs are down regulated by two principal factors, i.e. prolyl hydroxylases (PHDs) and von Hippel Lindau proteins (VHL). Tibetans have lived at 3,000-5,000 meters for approximately 20,000 years and have acquired a number of beneficial genetic adaptations which appear to prevent negative responses to hypoxia at high-altitude. Deciphering these genetic changes is crucial to improve our understanding of the underlying hypoxia-mediated response mechanisms and to develop targeted therapies. We recently identified the first Tibetan-specific mutation, PHD2D4E, caused by a missense mutation (rs186996510) in EGLN1. PHD2D4E has an allelic frequency of ~85% in Tibetans and a low Km for oxygen, accounting for the protection of Tibetans from high-altitude polycythemia. Other effects of PHD2D4E on HIF-regulated pathophysiology remain to be delineated. A 77% GC-rich area surrounds rs186996510, resulting in a low success rate of detecting the mutation by Sanger sequencing or next-generation sequencing. PHD2D4E was unreported in published whole-genome analyses of Tibetans (Xin Yi et. al. Science 2010).Here we describe a high-resolution melting assay of a small PCR product for targeted genotyping of rs186996510. The single base-pair change (G to C) is visualized by melting small amplicons in the presence of a fluorescent DNA-binding dye. Heterozygotes are differentiated from homozygous genotypes by a pronounced change in the shape of the melting curve caused by the formation of heteroduplexes. However, wild type and homozygous variants are difficult to distinguish by melting alone, and require an additional step of a second melting analysis after mixing with known wild type DNA. Upon melting these mixtures, homozygotes appear as heterozygous melting curves, while wild type genotypes will remain wild type (Figure 1).We developed and validated a high resolution melting assay for rapid genotyping of PHD2D4E suitable for population and disease association studies. In our ongoing analyses, we genotyped DNA from over 300 Tibetans residing at sea level, 1300 meters, 1730-2300 meters and 4320 meters, and are correlating the allelic frequency of PHD2D4E with hematocrit levels. The high resolution melting assay for genotyping PHD2D4E is a simple, accurate, rapid, and inexpensive approach to identify SNP-targeted mutations, especially suitable for a large number of samples such as needed for population studies, without the expense and time required for sequencing studies. [Display omitted] DisclosuresWittwer:BioFire Diagnostics: Aspects of melting analysis Patents & Royalties, Membership on an entity’s Board of Directors or advisory committees, Research Funding.

MiR-99a directly targets the mTOR signalling pathway in breast cancer side population cells.

miR-99a has been reported to function as a tumour suppressor in breast cancer. However, its role in the regulation of breast cancer stem cell (CSC) phenotype has up to now remained unknown. In this study, we isolated the side population (SP) cells by staining cultured MCF-7 and MDA-MB-231 cells with fluorescent DNA-binding dye Hoechst 33342, then by flow cytometric sorting. Next, we detected expression of miR-99a in the SP cells compared to non-SP cells using real-time PCR, and explored effects of miR-99a on the CSC phenotype of the breast cancer cells, including sphere formation, self-renewal, tumourigenicity and cell migratory capability. We found that expression of miR-99a was down-regulated in the SP cells compared to non-SP cells. Restoration of expression of miR-99a inhibited cell migration and invasion, reduced sphere formation of breast SP cells in vitro, and suppressed tumour growth in vivo. Finally, bioinformatic prediction suggested the oncogene, mammalian target of rapamycin (mTOR) - a downstream effector of the PI3K/AKT signalling pathway, was a target gene of miR-99a in SP cells. Further, quantitative RT-PCR and western blot assays identified that overexpression of miR-99a suppressed expression of mTOR and its downstream gene, HIF-1α. Collectively, these data suggest that miR-99a reversed the breast cancer malignant CSC phenotype, probably by targeting the mTOR signalling pathway.

In situ immunohistochemical detection of intracellular Mycoplasma salivarium in the epithelial cells of oral leukoplakia.

BackgroundMycoplasmas are the smallest free-living organisms; Mycoplasma salivarium and Mycoplasma orale are the most common species isolated from the oropharynx. Oral leukoplakia is the most prevalent potentially malignant disorder of the oral mucosa; its etiology has not been defined. Our previous study with DNA-binding fluorescent dye suggested the presence of mycoplasmas in the epithelial cells of leukoplakia tissue.ObjectiveOur aim was to detect M. salivarium in the epithelial cells of leukoplakia by immunohistochemistry.DesignWe produced a polyclonal antibody (PAb) reactive to Mycoplasma by injecting a rabbit with M. salivarium cells (ATCC 23064) mixed with complete Freund's adjuvant and a monoclonal antibody specific to M. salivarium by injecting M. salivarium cells (ATCC 23557) mixed with complete Freund's adjuvant into the footpads of a rat. Then, we attempted to detect M. salivarium in the epithelium of leukoplakia tissues by immunohistochemistry.ResultsWe obtained an antimycoplasma rabbit PAb reactive to all seven Mycoplasma species used in this study. Three hybridoma clones producing monoclonal antibodies specific to M. salivarium were obtained, and an M. salivarium-specific monoclonal antibody, designated 7-6H, was established. Immunohistochemistry with these antibodies revealed M. salivarium in the epithelial cells of leukoplakia with hyperplasia and hyperkeratosis on histology. PCR and sequencing verified the presence of M. salivariumDNA in the epithelial cells of leukoplakia.ConclusionIntracellular M. salivarium was identified in the epithelial cells of leukoplakia.

Abstract 478: Single-Cell Isolation and Analysis of Viable Proliferating Macrophages From Atherosclerotic Plaques

INTRODUCTION: We have recently shown that the proliferation of macrophages within the atherosclerotic plaque, rather than the recruitment of new monocytes from the blood is the key driver of plaque growth in established atherosclerosis. The study of proliferating macrophages has been hampered by the lack of a technique for identifying proliferating macrophages and isolating them from the atherosclerotic plaque in a viable state. OBJECTIVE: Develop and validate a process for identifying proliferating macrophages in atherosclerotic lesions, and isolating them as single, viable cells, then perform a molecular characterization of these cells. METHODS: ApoE-/- and LDLR-/- mice were fed a diet high in fat and cholesterol. Atherosclerotic aortas were collected, and cells were isolated by mechanical and enzymatic dissociation. Cells were stained using fluorescent markers and DNA-binding dyes, then analyzed and isolated by fluorescence-activated cell sorting. RESULTS: Mincing and enzymatic digestion in collagenase I, collagenase XI, DNAse, and hyaluronidase optimally isolates macrophages from aortic explants. Cell-surface staining with fluorescent antibodies against B220, F4/80, Ly6c, MHC II, and CD11b allows identification of lesional macrophages. DNA staining with Vybrant DyeCycle Violet allows identification of proliferating cells. Combined, these processes allow for the FACS-based sorting of viable proliferating macrophages from atherosclerotic plaque, and subsequent RNA or protein analysis, or culture. CONCLUSIONS: The process described here allows the first-ever viable isolation of proliferating macrophages from atherosclerotic plaques. This will permit the investigation of molecular targets to interfere with the pathogenesis of atherosclerosis.

Autophagy Induced by Calcium Phosphate Precipitates Targets Damaged Endosomes

Calcium phosphate precipitates (CPPs) form complexes with DNA, which enter cells via endocytosis. Under this condition CPPs induce autophagy via the canonic autophagy machinery. Here we showed that CPP-induced autophagy was also dependent on endocytosis as the process was significantly inhibited by methyl-β-cyclodextrin and dynasore, which suppress clathrin-dependent endocytosis. Consistently, CPP treatment triggered the formation of filipin-positive intracellular vesicles whose membranes are rich in cholesterol. Unexpectedly, these vesicles were also positive for galectin 3, suggesting that they were damaged and the membrane glycans became accessible to galectins to bind. Endosome damage was caused by endocytosis of CPPs and was reversed by calcium chelators or by endocytosis inhibitors. Notably, CPP-induced LC3-positive autophagosomes were colocalized with galectin 3, ubiquitin, and p62/SQSTM1. Inhibition of galectin 3 reduced p62 puncta and autophagosome formation. Knockdown of p62 additionally inhibited the colocalization of autophagosomes with galectins. Furthermore, most of the galectin 3-positive vesicles were colocalized with Rab7 or LAMP1. Agents that affect endosome/lysosome maturation and function, such as bafilomycin A1, also significantly affected CPP-induced tubulovesicular autophagosome formation. These findings thus indicate that endocytosed CPPs caused endosome damage and recruitment of galectins, particularly at the later endosome stage, which led to the interaction of the autophagosomal membranes with the damaged endosome in the presence of p62.

“Mega” cytometry for a “mega” challenging cell type

“Mega” cytometry for a “mega” challenging cell type