Joanne Lannigan

Imaging flow cytometry elucidates limitations of microparticle analysis by conventional flow cytometry

Microparticles (MPs) are submicron vesicles released from cell membranes in response to activation, cell injury, or apoptosis. The clinical importance of MPs has become increasingly recognized, although no standardized method exists for their measurement. Flow cytometry (FCM) is the most commonly used technique, however, because of the small size of MPs, and the limitations of current FCM instrumentation, accurate identification is compromised by this methodology. We decided to investigate whether the use of FCM combined with imaging, such as is possible with the ImagestreamX imaging FC (ISX), would be a more sensitive approach to characterizing MPs. Combining FCM with imaging eliminates some of the limitations demonstrated by conventional FCM, whereas also providing morphological confirmation and the ability to distinguish true single events from aggregates and cell debris. The detection limit of standard nonspecialized FCM is suboptimal when compared to ISX. Evaluating MPs below 0.200 µm and sizing remain a challenge as some MPs remain below the detection limit of ISX. Standardized calibrators, that more closely reflect the physical characteristics of MPs, need further development.

Murine lung eosinophil activation and chemokine production in allergic airway inflammation

Eosinophils play important roles in asthma and lung infections. Murine models are widely used for assessing the functional significance and mechanistic basis for eosinophil involvements in these diseases. However, little is known about tissue eosinophils in homeostasis. In addition, little data on eosinophil chemokine production during allergic airway inflammation are available. In this study, the properties and functions of homeostatic and activated eosinophils were compared. Eosinophils from normal tissues expressed costimulation and adhesion molecules B7-1, B7-2 and ICAM-1 for Ag presentation but little major histocompatibility complex (MHC) class II, and were found to be poor stimulators of T-cell proliferation. However, these eosinophils expressed high levels of chemokine mRNA including C10, macrophage inflammatory protein (MIP)-1α, MIP-1γ, MIP-2, eotaxin and monocyte chemoattractant protein-5 (MCP-5), and produced chemokine proteins. Eosinophil intracellular chemokines decreased rapidly with concomitant surface marker downregulation upon in vitro culturing consistent with piecemeal degranulation. Lung eosinophils from mice with induced allergic airway inflammation exhibited increased chemokines mRNA expression and chemokines protein production and upregulated MHC class II and CD11c expression. They were also found to be the predominant producers of the CCR1 ligands CCL6/C10 and CCL9/MIP-1γ in inflamed lungs. Eosinophil production of C10 and MIP-1γ correlated with the marked influx of CD11bhigh lung dendritic cells during allergic airway inflammation and the high expression of CCR1 on these dendritic cells (DCs). The study provided baseline information on tissue eosinophils, documented the upregulation of activation markers and chemokine production in activated eosinophils, and indicated that eosinophils were a key chemokine-producing cell type in allergic lung inflammation.

Optimization of yeast cell cycle analysis and morphological characterization by multispectral imaging flow cytometry

Budding yeast Saccharoymyces cerevisiae is a powerful model system for analyzing eukaryotic cell cycle regulation. Yeast cell cycle analysis is typically performed by visual analysis or flow cytometry, and both have limitations in the scope and accuracy of data obtained. This study demonstrates how multispectral imaging flow cytometry (MIFC) provides precise quantitation of cell cycle distribution and morphological phenotypes of yeast cells in flow. Cell cycle analysis of wild‐type yeast, nap1Δ, and yeast overexpressing NAP1, was performed visually, by flow cytometry and by MIFC. Quantitative morphological analysis employed measurements of cellular length, thickness, and aspect ratio in an algorithm to calculate a novel feature, bud length. MIFC demonstrated reliable quantification of the yeast cell cycle compared to morphological and flow cytometric analyses. By employing this technique, we observed both the G2/M delay and elongated buds previously described in the nap1Δ strain. Using MIFC, we demonstrate that overexpression of NAP1 causes elongated buds yet only a minor disruption in the cell cycle. The different effects of NAP1 expression level on cell cycle and morphology suggests that these phenotypes are independent. Unlike conventional yeast flow cytometry, MIFC generates complete cell cycle profiles and concurrently offers multiple parameters for morphological analysis.

CD44 deficiency attenuates chronic murine ileitis.

BACKGROUND & AIMS Lymphocyte recruitment to sites of inflammation requires the sequential engagement of adhesion molecules and chemokine receptors. In the current studies we analyzed the role of CD44 for the development of chronic small-intestinal inflammatory infiltrates in vivo. METHODS By using a tumor necrosis factor (TNF)-driven model of chronic ileitis (ie, B6.129P-TNF(DeltaAU-rich element [ARE])) that recapitulates many features of Crohns disease, we noticed dynamic changes in the expression and functional state of CD44 and its ligand hyaluronan via enzyme-linked immunosorbent assay, real-time reverse-transcription polymerase chain reaction, immunohistochemistry, and flow cytometry. In addition, we assessed the role of lymphocyte populations during induction of ileitis through adoptive transfer studies, and generated CD44-deficient TNFDeltaARE mice to assess the role of CD44 for development of ileitis. RESULTS Soluble hyaluronan levels and expression of hyaluronan synthase-1 were increased in TNFDeltaARE mice. This coincided with increased expression of CD44 (including variant 7) and reactivity towards hyaluronan on CD4(+) T cells. CD44 was spatially colocalized with the gut-homing integrin alpha(4)beta(7), spatially linking lymphocyte rolling with arrest. These cells had an effector phenotype because they lacked L-selectin and a higher proportion in diseased mice produced TNF and interleukin-2 compared with wild-type littermates. Lastly, CD4(+) but not CD8(+) T cells conferred ileitis to RAG(-/-) recipients and deficiency of one or both alleles of the CD44 gene resulted in attenuation of the severity of ileitis in TNFDeltaARE mice. CONCLUSIONS Our findings support an important role of CD44 expressed by CD4(+) and CD8(+) for development of ileitis mediated by TNF overproduction.

Practical issues in high-speed cell sorting.

Modern flow cytometric cell sorters are all capable of so‐called “high‐speed sorting.” However, there is confusion about exactly how fast a “high‐speed” cell sorter can sort cells. There are many considerations in achieving the fastest sorting speed, as well as the highest quality sort results—cell recovery, purity, and functionality. This requires the same considerations required for “slow‐speed sorting”; however, a more precise implementation is required for high‐speed sorting. The modern cell sorters enable high‐speed sorting because of advances in high‐speed electronics and data processing. We discuss the practical considerations of high‐speed sorting in terms of the theory and practical aspects of the mechanical and software components of sorting, statistics of sorting, cell preparation and viability, instrument setup, sort strategies, and biosafety. Curr. Protoc. Cytom. 51:1.24.1‐1.24.30.

An improved method for differentiating cell-bound from internalized particles by imaging flow cytometry

Recognition, binding, internalization, and elimination of pathogens and cell debris are important functions of professional as well as non-professional phagocytes. However, high-throughput methods for quantifying cell-associated particles and discriminating bound from internalized particles have been lacking. Here we describe a protocol for using imaging flow cytometry to quantify the attached and phagocytosed particles that are associated with a population of cells. Cells were exposed to fluorescent particles, fixed, and exposed to an antibody of a different fluorophore that recognizes the particles. The antibody is added without cell permeabilization, such that the antibody only binds extracellular particles. Cells with and without associated particles were identified by imaging flow cytometry. For each cell with associated particles, a spot count algorithm was employed to quantify the number of extracellular (double fluorescent) and intracellular (single fluorescent) particles per cell, from which the percent particle internalization was determined. The spot count algorithm was empirically validated by examining the fluorescence and phase contrast images acquired by the flow cytometer. We used this protocol to measure binding and internalization of the bacterium Neisseria gonorrhoeae by primary human neutrophils, using different bacterial variants and under different cellular conditions. The results acquired using imaging flow cytometry agreed with findings that were previously obtained using conventional immunofluorescence microscopy. This protocol provides a rapid, powerful method for measuring the association and internalization of any particle by any cell type.

Does FACS perturb gene expression

Fluorescence activated cell sorting is the technique most commonly used to separate primary mammary epithelial sub‐populations. Many studies incorporate this technique before analyzing gene expression within specific cellular lineages. However, to our knowledge, no one has examined the effects of fluorescence activated cell sorting (FACS) separation on short‐term transcriptional profiles. In this study, we isolated a heterogeneous mixture of cells from the mouse mammary gland. To determine the effects of the isolation and separation process on gene expression, we harvested RNA from the cells before enzymatic digestion, following enzymatic digestion, and following a mock FACS sort where the entire cohort of cells was retained. A strict protocol was followed to minimize disruption to the cells, and to ensure that no subpopulations were enriched or lost. Microarray analysis demonstrated that FACS causes minimal disruptions to gene expression patterns, but prior steps in the mammary cell isolation process are followed by upregulation of 18 miRNAs and rapid decreases in their predicted target transcripts.

Optimized Protocol for the Isolation of Spleen-Resident Murine Neutrophils

Neutrophils are an important cellular component of the innate immune system that provides immediate protection to the host from infection. Neutrophil infiltration into inflamed peripheral tissues during infection is beneficial for immunity through phagocytosis of microbes, the release of antimicrobial factors, and secretion of proinflammatory cytokines. Recent reports further suggest that spleen‐infiltrating neutrophils play a role in the adaptive immune response by providing survival signals to B cells. However, neutrophils may have detrimental effects on immunity in inflammatory diseases where their recruitment to lymphoid tissues and activation occur abnormally. To determine the contribution of neutrophils that reside in secondary lymphoid tissues to adaptive immunity, direct evaluation of the functional properties of tissue‐resident neutrophils is required. We have developed a modified magnetic bead isolation approach for purifying neutrophils from inflamed spleens of autoimmune‐prone mice by negative selection. Using this approach, we yielded neutrophils with greater than 90% purity without compromising cell viability. Equally important, the isolation procedure had little effect on the activation of neutrophils and did not impair phagocytic function. Thus, isolation of spleen‐resident neutrophils by this optimized approach could be useful for interrogating the functional role of murine neutrophils in normal and abnormal immune responses.

Imaging Flow Cytometry for the Characterization of Extracellular Vesicles.

Extracellular Vesicles (EVs) are potent bio-activators and inter-cellular communicators that play an important role in both health and disease. It is for this reason there is a strong interest in understanding their composition and origin, with the hope of using them as important biomarkers or therapeutics. Due to their very small size, heterogeneity, and large numbers there has been a need for better tools to measure them in an accurate and high throughput manner. While traditional flow cytometry has been widely used for this purpose, there are inherent problems with this approach, as these instruments have traditionally been developed to measure whole cells, which are orders of magnitude larger and express many more molecules of identifying epitopes. Imaging flow cytometry, as performed with the ImagestreamX MKII, with its combination of increased fluorescence sensitivity, low background, image confirmation ability and powerful data analysis tools, provides a great tool to accurately evaluate EVs. We present here a comprehensive approach in applying this technology to the study of EVs.

Systems analysis of the transcriptional response of human ileocecal epithelial cells to Clostridium difficile toxins and effects on cell cycle control

BackgroundToxins A and B (TcdA and TcdB) are Clostridium difficiles principal virulence factors, yet the pathways by which they lead to inflammation and severe diarrhea remain unclear. Also, the relative role of either toxin during infection and the differences in their effects across cell lines is still poorly understood. To better understand their effects in a susceptible cell line, we analyzed the transciptome-wide gene expression response of human ileocecal epithelial cells (HCT-8) after 2, 6, and 24 hr of toxin exposure.ResultsWe show that toxins elicit very similar changes in the gene expression of HCT-8 cells, with the TcdB response occurring sooner. The high similarity suggests differences between toxins are due to events beyond transcription of a single cell-type and that their relative potencies during infection may depend on differential effects across cell types within the intestine. We next performed an enrichment analysis to determine biological functions associated with changes in transcription. Differentially expressed genes were associated with response to external stimuli and apoptotic mechanisms and, at 24 hr, were predominately associated with cell-cycle control and DNA replication. To validate our systems approach, we subsequently verified a novel G1/S and known G2/M cell-cycle block and increased apoptosis as predicted from our enrichment analysis.ConclusionsThis study shows a successful example of a workflow deriving novel biological insight from transcriptome-wide gene expression. Importantly, we do not find any significant difference between TcdA and TcdB besides potency or kinetics. The role of each toxin in the inhibition of cell growth and proliferation, an important function of cells in the intestinal epithelium, is characterized.