Brian E. Hall

Distinguishing modes of cell death using the ImageStream multispectral imaging flow cytometer.

Here we demonstrate the ability of the ImageStream® 100 Multispectral Imaging Cytometer to discriminate between live, necrotic, and early and late apoptotic cells, using unique combinations of photometric and morphometric features.

Extended depth of field imaging for high speed object analysis

Fluoresence microscopy is an extremely useful tool to analyze the intensity, location and movement of fluorescently tagged molecules on, within or between cells. However, the technique suffers from slow image acquisition rates and limited depth of field. Confocal microscopy addresses the depth of field issue via “optical sectioning and reconstruction”, but only by further reducing the image acquisition rate to repeatedly scan the cell at multiple focal planes. In this paper we describe a technique to perform high speed, extended depth of field (EDF) imaging using a modified ImageStream® system whereby high resolution, multimode imagery from thousands of cells is collected in less than a minute with focus maintained over a 16 μm focal range.

C1q Deficiency Leads to the Defective Suppression of IFN-α in Response to Nucleoprotein Containing Immune Complexes

Almost all humans with homozygous deficiency of C1q develop systemic lupus erythematosus (SLE). The precise cellular mechanism(s) by which C1q prevents the development of SLE remains unclear. In this study, we tested the role of C1q in the regulation of IFN-α induced by immune complexes (ICs) in vitro, as well as the consequences of lack of C1q in vivo. Our experiments revealed that C1q preferentially promotes the binding of SLE ICs to monocytes rather than plasmacytoid dendritic cells, but this inhibition was not due to the induction of inhibitory soluble factors. The presence of C1q also altered the trafficking of ICs within monocytes such that ICs persisted in early endosomes. In patients with C1q deficiency, serum and cerebrospinal fluid levels of IFN-α and IFN-γ–inducible protein-10 levels were elevated and strongly correlated with Ro autoantibodies, demonstrating the clinical significance of these observations. These studies therefore associate C1q deficiency with defective regulation of IFN-α and provide a better understanding of the cellular mechanisms by which C1q prevents the development of IC-stimulated autoimmunity.

Sensitivity measurement and compensation in spectral imaging

The ImageStream system combines advances in CCD technologies with a novel optical architecture for high sensitivity and multispectral imaging of cells in flow. The sensitivity and dynamic range as well as a methodology for spectral compensation of imagery is presented.

Conventional apoptosis assays using propidium iodide generate a significant number of false positives that prevent accurate assessment of cell death.

The advent of flow cytometry-based applications has significantly impacted the study of cellular apoptosis. Propidium iodide (PI) is a commonly used viability stain in these studies. Unfortunately, we find that conventional Annexin V/PI protocols lead to a significant number of false positive events (up to 40%), which are associated with PI staining of RNA within the cytoplasmic compartment. Both primary cells and cell lines are affected, with large cells (nuclear: cytoplasmic ratios <0.5) showing the highest occurrence. This distribution spans a wide range of animal models including mice, swine, avian, and teleost fish and potentially affects up to 1016 out of 1019 of peer-reviewed papers published in this area since 1995. We show that the primary ramifications from these findings relate to cells experiencing changes in RNA content. Virally infected cells, for example, are qualified as undergoing apoptosis in response to infection based on conventional staining protocols; in fact, these cells are alive and actively producing viral RNA that can serve to produce additional infectious viral particles. Based on our observations we propose a modified protocol, show that it overcomes previous drawbacks for this technique, and that it will allow for more accurate assessment of cell death across various platforms.

Macrophage activation differentially modulates particle binding, phagocytosis and downstream antimicrobial mechanisms.

Phagocytosis provides a critical first line of defense against invading pathogens. Engagement of particles through receptor-mediated binding precedes internalization and induction of cellular antimicrobial responses. Phagocytes have the capacity to differentially regulate binding and internalization processes through changes in their receptor profile and modulation of downstream events. This is necessary for the intricate control of phagocytic antimicrobial responses. Several methods are available for evaluation of phagocytosis. Unfortunately, none allow for accurate quantitation of both binding and internalization events. To overcome these limitations, we have developed a novel phagocytosis assay based on a multi-spectral imaging flow cytometry platform. This assay discriminates between internalized and surface-bound particles in a statistically robust manner and allows multi-parametric analysis of phagocytosis and downstream anti-microbial responses. We also devised a novel approach for examination of phagolysosome fusion, which provides an improved capacity for quantitative assessment of phagolysosome fusion in mixed populations of intact cells. Importantly, our approaches are likely amenable to a broad range of comparative model systems based on our examination of murine RAW 264.7 cells and a goldfish primary kidney macrophage (PKM) model system. The latter allowed us to examine the evolutionary conservation of phagocytic antimicrobial responses in a lower vertebrate model. While it has been previously reported that mixed populations of these macrophage cultures are phagocytic, it remained unclear if sub-populations within them contributed differentially to this activity. In accordance with higher vertebrate models, we found that differentiation along the macrophage pathway leads to an increased capacity for phagocytosis in goldfish PKM. Interestingly, cellular activation differentially regulated particle internalization in PKM monocyte and mature macrophage subsets. We also found differential regulation of phagolysosome fusion and downstream production of reactive oxygen intermediates (ROI). The temporal activation of specific phagocytic antimicrobial responses at distinct stages of PKM differentiation suggests specialization within the macrophage compartment early in evolution, geared to meet specific host immunity requirements within specialized niches.

Cutting Edge: FADD Is Not Required for Antigen Receptor-Mediated NF-κB Activation

Recently, it has been demonstrated that stimulated T cells bearing defects in caspase-8 fail to promote nuclear shuttling of NF-κB complexes. Such cells display strikingly similar proliferative and survival defects as T cells lacking Fas-associated death domain protein (FADD) function. We characterized NF-κB signaling in T cells bearing a dominant-negative FADD transgene (FADDdd). Whereas FADDdd T cells displayed proliferative defects following activation, these were not a consequence of aberrant NF-κB signaling, as measured by IKK/IκB phosphorylation and IκB degradation. There were no appreciable defects in nuclear translocation of p65/Rel using ImageStream, a flow-based imaging cytometer. Pretreatment with benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone, a potent caspase inhibitor, also failed to impede canonical NF-κB signaling. Secretion of IL-2 and up-regulation of various activation markers occurred normally. Thus, FADD does not play an essential role in NF-κB activation, suggesting an alternative route by which this adaptor promotes the clonal expansion of T cells.

Small-molecule suppressors of Candida albicans biofilm formation synergistically enhance the antifungal activity of amphotericin b against clinical Candida isolates

A new class of fungal biofilm inhibitors represented by shearinines D (3) and E (4) were obtained from a Penicillium sp. isolate. The inhibitory activities of 3 and 4 were characterized using a new imaging flow-cytometer technique, which enabled the rapid phenotypic analysis of Candida albicans cell types (budding yeast cells, germ tube cells, pseudohyphae, and hyphae) in biofilm populations. The results were confirmed by experimental data obtained from three-dimensional confocal laser scanning microscopy and 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) assays. These data indicate that 3 and 4 inhibited C. albicans biofilm formation by blocking the outgrowth of hyphae at a relatively late stage of biofilm development (IC50 = 8.5 and 7.6 μM, respectively). However, 3 and 4 demonstrated comparatively weak activity at disrupting existing biofilms. Compounds 3 and 4 also exhibited synergistic activities with amphotericin B against C. albicans and other clinical Candida isolates by enhancing the potency of amphotericin B up to 8-fold against cells in both developing and established biofilms. These data suggest that the Candida biofilm disruption and amphotericin B potentiating effects of 3 and 4 could be mediated through multiple biological targets. The shearinines are good tools for testing the potential advantages of using adjunctive therapies in combination with antifungals.

CD81 is essential for the re-entry of hematopoietic stem cells to quiescence following stress-induced proliferation via deactivation of the Akt pathway.

A protein that is thought to orchestrate the distribution of other signaling molecules on the cell membrane, CD81, is critical to maintaining the functional integrity of hematopoietic stem cells during their regeneration.

Exploring the activated adipogenic niche: Interactions of macrophages and adipocyte progenitors

Adult adipose tissue contains a large supply of progenitors that can renew fat cells for homeostatic tissue maintenance and adaptive growth or regeneration in response to external challenges. However, the in vivo mechanisms that control adipocyte progenitor behavior are poorly characterized. We recently demonstrated that recruitment of adipocyte progenitors by macrophages is a central feature of adipose tissue remodeling under various adipogenic conditions. Catabolic remodeling of white adipose tissue by β3-adrenergic receptor stimulation requires anti-inflammatory M2-polarized macrophages to clear dying adipocytes and to recruit new brown adipocytes from progenitors. In this Extra Views article, we discuss in greater detail the cellular elements of adipogenic niches and report a strategy to isolate and characterize the subpopulations of macrophages and adipocyte progenitors that actively participate in adrenergic tissue remodeling. Further characterization of these subpopulations may facilitate identification of new cellular targets to improve metabolic and immune function of adipose tissue.