Detection of regulatory T cells in germinal centers using FDMM.

<p>Mice were immunized i.p. with a mixture of polyI:C and ovalbumin to induce formation of germinal centers. Ten days later the spleens were harvested, sectioned and multi-immunolabeled with antibodies against IgD-expressing B cells (anti-IgD, red), follicular dendritic cells (anti-MFG-E8, cyan), CD4⁺ T cells (anti-CD4, green), the transcription factor Foxp3 (anti-Foxp3, white), B cells (anti-B220, not shown) and nuclei (DAPI, blue). <i>(A)</i> Detection of a germinal center. The picture shows the expression of IgD (red, negative marker) and MGF-E8 (cyan, positive marker). <i>(B)</i> Detection of regulatory T cells (CD4⁺Foxp3⁺) in a germinal center (arrow) and outside a germinal center (arrowhead). All channels are presented individually in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0119499#pone.0119499.s004" target="_blank">S4 Fig</a>. Scale bar, 50 μm.</p

Filter-Dense Multicolor Microscopy

<div><p>Immunofluorescence microscopy is a unique method to reveal the spatial location of proteins in tissues and cells. By combining antibodies that are labeled with different fluorochromes, the location of several proteins can simultaneously be visualized in one sample. However, because of the risk of bleed-through signals between fluorochromes, standard multicolor microscopy is restricted to a maximum of four fluorescence channels, including one for nuclei staining. This is not always enough to address common scientific questions. In particular, the use of a rapidly increasing number of marker proteins to classify functionally distinct cell populations and diseased tissues emphasizes the need for more complex multistainings. Hence, multicolor microscopy should ideally offer more channels to meet the current needs in biomedical science. Here we present an enhanced multi-fluorescence setup, which we call Filter-Dense Multicolor Microscopy (FDMM). FDMM is based on condensed filter sets that are more specific for each fluorochrome and allow a more economic use of the light spectrum. FDMM allows at least six independent fluorescence channels and can be applied to any standard fluorescence microscope without changing any operative procedures for the user. In the present study, we demonstrate an FDMM setup of six channels that includes the most commonly used fluorochromes for histology. We show that the FDMM setup is specific and robust, and we apply the technique on typical biological questions that require more than four fluorescence microscope channels.</p></div

Detection of lipid-loaded dendritic cells in an atherosclerotic lesion using FDMM.

<p>Atherosclerotic lesions were induced in the carotid artery of LDLr^−/− mice. The carotid arteries were harvested, sectioned and multi-immunolabeled with antibodies against lipid droplets (anti-perilipin 2, red), smooth muscle cells (anti-SM α-actin, cyan), antigen presenting cells (anti-MHC class II, green, upper left), CD11c (anti-CD11c, green, lower left), and CD11b (anti-CD11b, green, lower right). Nuclei were stained with DAPI (blue). Arrows depict cells that are positive for MHC class II, CD11c and perilipin 2, and negative/dim for CD11b. The small arrowheads indicate arterial elastin fibers (laminae), which autofluoresce in the 425 channel. All six channels are presented individually in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0119499#pone.0119499.s005" target="_blank">S5 Fig</a>. Scale bar, 100 μm.</p

Examples of fluorochromes that fit in the FDMM setup.

<p>* fluorochromes that were tested in the FDMM setup</p><p>Examples of fluorochromes that fit in the FDMM setup.</p

The strategy and specificity of FDMM.

<p><i>(A)</i> Example of a condensed filter set compared with a standard filter set (Carl Zeiss, filter set #38) for the 488 channel. The curves show the excitation (blue line) and emission (red line) light spectra of the fluorochrome AF488. Blue rectangles, excitation filter interval; red rectangles, emission filter interval; vertical black line, beam splitter. <i>(B)</i> By the use of condensed filter sets, the density of fluorescence channels is increased in the FDMM setup compared with standard multicolor microscopy. <i>(C)</i> Each filter set in the FDMM setup is specific for its corresponding fluorochrome. Six tissue sections from a blood vessel were immunolabeled for CD31 (endothelium) with different fluorochromes or nuclei stained with DAPI. Each filter set (columns) received signals from only one fluorochrome (rows). Scale bar, 100 μm. Exposure times: DAPI channel (11 ms), 425 channel (30 ms), 488 channel (63 ms), Cy3 channel (20 ms), 594 channel (54 ms), PerCP channel (17 ms). Objective: x20/0.75.</p

Multicolor antibody array for mouse spleen using FDMM.

<p>Tissue sections from a wild type spleen (WT), a TNFα-receptor 1 knockout spleen (TNFaR1 KO), and a spleen from a CD11b-DTR mouse (CD11b-DTR) were multi-immunolabeled with antibodies against marginal zone macrophages (anti-CD169, cyan), dendritic cells (anti-CD11c, blue), B cells (anti-B220, red), CD4⁺ T cells (anti-CD4, green), and CD8⁺ T cells (anti-CD8, magenta). Nuclei were stained with DAPI (not shown). Arrow in TNFα-receptor 1 knockout spleen indicates weak marginal zone structure (cyan). Arrowhead in CD11b-DTR spleen indicates CD4⁺ T cells (green) within the B cell follicles. All six channels are presented individually in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0119499#pone.0119499.s006" target="_blank">S6 Fig</a>. Scale bar, 100 μm.</p

Six channel FDMM for subcellular staining of cultured NIH cells.

<p>Cultured NIH cells immunolabeled for different intracellular structures. Upper small pictures: Nuclei (DAPI channel, DAPI, 12 ms), Focal adhesions (425 channel, anti-vinculin, 90 ms), Lipid droplets (488 channel, anti-ADRP, 50 ms), Golgi (Cy3 channel, anti-syntaxin 5, 10 ms), F-actin cytoskeleton (594 channel, phalloidin, 17 ms), and Lyzosomes (PerCP channel, anti-LAMP 1, 46 ms). Scale bar, 40 μm. Objective x63/1.4 Oil DIC. Lower large picture: Merged image of all channels. Scale bar, 20 μm.</p

Fucosylated Molecules Competitively Interfere with Cholera Toxin Binding to Host Cells

Cholera toxin (CT) enters host intestinal epithelia cells, and its retrograde transport to the cytosol results in the massive loss of fluids and electrolytes associated with severe dehydration. To initiate this intoxication process, the B subunit of CT (CTB) first binds to a cell surface receptor displayed on the apical surface of the intestinal epithelia. While the monosialoganglioside GM1 is widely accepted to be the sole receptor for CT, intestinal epithelial cell lines also utilize fucosylated glycan epitopes on glycoproteins to facilitate cell surface binding and endocytic uptake of the toxin. Further, l-fucose can competively inhibit CTB binding to intestinal epithelia cells. Here, we use competition binding assays with l-fucose analogs to decipher the molecular determinants for l-fucose inhibition of cholera toxin subunit B (CTB) binding. Additionally, we find that mono- and difucosylated oligosaccharides are more potent inhibitors than l-fucose alone, with the LeY tetrasaccharide emerging as the most potent inhibitor of CTB binding to two colonic epithelial cell lines (T84 and Colo205). Finally, a non-natural fucose-containing polymer inhibits CTB binding two orders of magnitude more potently than the LeY glycan when tested against Colo205 cells. This same polymer also inhibits CTB binding to T84 cells and primary human jejunal epithelial cells in a dose-dependent manner. These findings suggest the possibility that polymeric display of fucose might be exploited as a prophylactic or therapeutic approach to block the action of CT toward the human intestinal epithelium

CTB binds to Le^X-carrying proteins in HL-60 cells.

<p><b>(A)</b> Histogram from flow cytometry analysis of CTB-binding to HL-60 cells following pre-treatment of the cells with AAL (10 μg/ml) or pre-treatment of CTB with sugars (50 mM). <b>(B)</b> gMFI of CTB binding to HL-60 cells cultured with the indicated inhibitors (*** = p<0.001 and ** = p<0.01). <b>(C-D)</b> Western blot using anti-CTB of HL-60 cells co-cultured with <b>(C)</b> (NB-DGJ) or <b>(D)</b> (benzyl-α-GalNAc, kifunensine, or 2F-Fuc) and the precursor sugar Ac4ManNDAz to enable UV-crosslinking between CTB and glycosylated structures. Representative of two independent experiments. <b>(E)</b> Western blot using anti-Le^X of HL-60 cells after incubation with CTB, lysis and immunoprecipitation with anti-CTB. One representative out of two independent experiments is shown.</p

GM1 ganglioside-independent intoxication by Cholera toxin

<div><p>Cholera toxin (CT) enters and intoxicates host cells after binding cell surface receptors via its B subunit (CTB). We have recently shown that in addition to the previously described binding partner ganglioside GM1, CTB binds to fucosylated proteins. Using flow cytometric analysis of primary human jejunal epithelial cells and granulocytes, we now show that CTB binding correlates with expression of the fucosylated Lewis X (Le^X) glycan. This binding is competitively blocked by fucosylated oligosaccharides and fucose-binding lectins. CTB binds the Le^X glycan <i>in vitro</i> when this moiety is linked to proteins but not to ceramides, and this binding can be blocked by mAb to Le^X. Inhibition of glycosphingolipid synthesis or sialylation in GM1-deficient C6 rat glioma cells results in sensitization to CT-mediated intoxication. Finally, CT gavage produces an intact diarrheal response in knockout mice lacking GM1 even after additional reduction of glycosphingolipids. Hence our results show that CT can induce toxicity in the absence of GM1 and support a role for host glycoproteins in CT intoxication. These findings open up new avenues for therapies to block CT action and for design of detoxified enterotoxin-based adjuvants.</p></div