6 research outputs found

    Effect of glucose and lactate on cellular ATP content in IPEC-1 cells after 1 h treatment.

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    <p>IPEC-1 cells were grown to confluence (7 d) and treated for 1 h with HBSS-based solutions. Supernatant was removed and ATP content was measured by a luminescence based assay. Statistical analysis was done by Kruskal-Wallis and Dunn's post-hoc test. Different superscripts indicate statistically significant (p<0.05) differences.</p

    Quantification of cytosol / nucleus NADH ratio in IPEC-1 cells after 1 h treatment with glucose and lactate without further challenge.

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    <p>Cellular autofluorescence of IPEC-1 cells was monitored at ex: 340 nm / em: 510 nm. <b>(A)</b> Change in spatial distribution of autofluorescence in response to Anti A (100 μM). <b>(B)</b> Cytosolic autofluorescence in response to complex III inhibitor Anti A (100 μM) and uncoupler FCCP (1 μM). <b>(C)</b> IPEC-1 cells were treated as indicated for 1 h. Glucose (gluc) concentration was 1 g/L if not otherwise indicated. Cells were treated with lactate (lac) 25 mM or increased gluc 4.5 g/L. Autofluorescence measured after 1 h and ratio of cytosolic (F<sub>cytosol</sub>) and nuclei (F<sub>nucleus</sub>) was calculated (ex: 340 nm / em: 510 nm) from individual cells without further stimulation. Statistical analysis was done by Kruskal-Wallis and Dunn's post-hoc test. Different superscripts indicate statistically significant (p<0.05) differences.</p

    Attachment and structural effects of lactobacilli on IPEC-1 cells.

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    <p><b>(A)</b> Three dimensional reconstruction of lactobacilli attached to IPEC-1 cells. <i>Lactobacillus amlylovorus</i> strain GRL1110 (arrows) was applied for 6 h on confluent IPEC-1 cells. The bacterial suspension was then carefully removed; cells and attached bacteria were fixed and stained with DAPI (nuclei, blue) and CM-Dil (plasma membrane, red). Confocal sections were reconstructed to obtain a 3D view. Note the thin, barely visible cytosolic layer above nucleus (*). (<b>B-H</b>) Actin structure of IPEC-1 cells treated with lactate and <i>Lactobacillus amylovorus</i> supernatants. IPEC-1 cells were cultured on glass, incubated as indicated and stained for F-actin (Phalloidin-PromoFluor 546) and nuclei (DAPI). (<b>B</b>) control buffer pH7, (<b>C</b>) Na-DL-lactate (25 mM, pH 7), (<b>D</b>) control buffer pH 4, (<b>E</b>) lactic acid pH 4. (<b>F-H</b>) supernatants of <i>Lactobacillus amylovorus</i> strains. (<b>F</b>) GRL1110, (<b>G</b>) GRL1112, (<b>H</b>) GRL1115. Predominantly basal F-actin fibre structure was reduced by lactic acid and bacterial supernatants (pH 4), (<b>I</b>) F-actin staining of freeze-section of porcine jejunal epithel. Note positive F-actin signal indicating the apical terminal web (arrowhead), the low F-actin signal at the lateral side and the faint signal in the basal region of the enterocytes (arrow). Bar: 5 μm.</p

    Effect of weaning on intestinal lactate concentration and lactobacilli load of piglets.

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    <p><b>(A)</b> Lactate concentration <b>(B)</b> <i>Lactobacillus</i> load and <b>(C)</b> pH in chyme of suckling and weaned piglets along the intestinal axis was measured. Statistical analysis was performed with the procedure “MIXED” (SAS, version 8) and comparison between pre- and postweaning using the t-test (p<0.01).</p

    Normalised Anti A triggered ROS generation in IPEC cells after lactate and glucose treatment.

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    <p><b>(A)</b> IPEC-1 cells were cultured on glass (7 d), incubated for 1 h in buffer (HBSS), placed on microscopic stage and challenged with Anti A in the presence of DHE. Mean fluorescence of nuclei (regions of interest, white circles) was monitored over time. <b>(B)</b> Quantification of nucleic fluorescence of ethidium cation (ethidium cation is the oxidation product of DHE) binding to nuclei. Anti A (10 / 100 μM) or control buffer was applied in presence of DHE 1.5 min after starting the measurement. <b>(C)</b> Anti A (100 μM) triggered superoxide generation rate (DHE oxidation) of IPEC-1 and IPEC-J2 cells after 1 h treatment (39°C). Cells were treated with lactate (lac, 25 mM); lac + inhibitor of monocarboxylate transporter (MCT inhibitor, ARC155858, 10 μM); increased glucose (gluc, 4.5 g/L) or glycolysis inhibitor 2-Deoxyglucose (2DG, 10 mM). Glucose concentration was 1 g/L if not otherwise indicated. Slope of fluorescence increase of individual cell nuclei was calculated and normalised to the control. Statistical analysis was done by Kruskal-Wallis and Dunn's post-hoc test. Different superscripts indicate statistically significant (p<0.05) differences.</p

    Visualisation of adherence of bacteria on IPEC-1 cells.

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    <p>Three different strains of <i>Lactobacillus amylovorus</i> (GRL1110 (<b>B</b>); GRL1112 (<b>C</b>); GRL1115 (<b>D</b>)) were applied on confluent IPEC-1 cells at the cell:bacteria ratio 1:1000 for 6 h in cell culture medium. Bacterial suspensions were removed, cells and attached bacteria were fixed and stained with DAPI. Bacterial DNA indicates a different degree of adherence of bacteria to epithelial cell surface (<b>B-D</b>) in comparison to non-treated (<b>A</b>) cells. Representative micrographs of three independent experiments are shown. Bar: 5 μm.</p
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