Journal: bioRxiv

Article Title: Differential translocation of bacteriophages across the intestinal barrier in health and Crohn’s disease

doi: 10.1101/2024.09.17.613249

Figure Lengend Snippet: (A) Overview of the experimental setup. (B) Titration of T4, M13 and ΦX174 phages associated to Caco-2/TC7 cells (cell) and translocated through the monolayer (basal) after 24 h of incubation. The input refers to the phages initially added to the apical compartment. A minimum of four independent experiments [n ≥ 4] were performed. The table displays the percentage of phages associated to cells and translocated across Caco-2/TC7, normalized to the input. For details on quantification and cut-off criteria, see the Method section. (C) Images of CF488-labeled M13 phages interacting with Caco-2/TC7 cells following a 24 h of incubation. Examples of High+, Medium+ and Low+ fields are shown in insets, corresponding to different fluorescence levels of Caco-2/TC7 positive for CF488-labeled M13 phages. (D) Quantification of the percentage of Caco-2/TC7 ceils positive for CF488-labeled Ml3 phages, as shown in panel (C). Cells were categorized based on the fluorescence intensity of phages from analysis of 10 randomly selected images. (E) YZ views of CF488-labeled M13 with Caco-2/TC7 cells following 24 h of incubation. (F) Images (XY and YZ views) of CF488-labeled M13 phages combined with immunofluorescence stainings of tricellulin (tricellular tight junction), Zonula Occludens-1 (ZO-1, tight junction) and E-cadherin (adherens junction) after 24 h of incubation. In all fluorescence images, M13 phages are covalently conjugated with CF488 fluorophore (green), actin is labeled with Alexa A546-phalloidin (red), and nuclei with DAPI (blue). White arrows indicate phages colocalizing with actin (C), Tricellulin (F), ZO-1 (F), and E-cadherin (F) or internalized within cells (E). Scale bar, 10 pm. Data are represented as mean ± SEM.

Article Snippet: An aliquot of 1 µmol CF™ 488A succinimidyl ester (CF488-SE; Biotium) was dissolved in 100 µL anhydrous DMSO to obtain a 10 mM CF488-SE solution.

Techniques: Titration, Incubation, Labeling, Fluorescence, Immunofluorescence

Journal: bioRxiv

Article Title: Differential translocation of bacteriophages across the intestinal barrier in health and Crohn’s disease

doi: 10.1101/2024.09.17.613249

Figure Lengend Snippet: (A) Overview of the experimental setup. (B) Titration of T4, M13, and ΦX174 in the basal compartment (basal) after 1 h and 24 h of incubation. The input refers to the phages initially added to the apical compartment. The table displays the percentage of translocated phages across HUVECs, normalized to the input. (C) Images of CF488-labeled M13 phages with HUVECs cells after 30 min, 1 h, 4 h, and 24 h of incubation. Actin was labeled with Alexa A546-phalloidin (red), and nuclei with DAPI (blue). The YZ view corresponds to 24 h of incubation. (D) Quantification of the M13-CF488 fluorescence shown in panel (C). Data represent mean values ± SEM obtained from the analysis of 5 randomly selected images. (E) Images of CF4884abeled M13 phages, and Cy3-labeled transferrin in HUVECs cells after 4 h, at 37°C, and 4°C. (F) Quantification of the red fluorescence area (transferrin), and green fluorescence area (M13 phages) in HUVECs following 4 h, at 37°C, and 4°C, shown in panel (E). Data represent mean values ± SEM obtained from analysis of 5 randomly selected images. (G) Images of CF488-labeled M13 phages, and Lysotracker Deep Red in HUVECs cells after 4 h, and 24 h. The YZ view corresponds to 24 h of incubation. Scale bar, 10 pm.

Article Snippet: An aliquot of 1 µmol CF™ 488A succinimidyl ester (CF488-SE; Biotium) was dissolved in 100 µL anhydrous DMSO to obtain a 10 mM CF488-SE solution.

Techniques: Titration, Incubation, Labeling, Fluorescence

Journal: bioRxiv

Article Title: Differential translocation of bacteriophages across the intestinal barrier in health and Crohn’s disease

doi: 10.1101/2024.09.17.613249

Figure Lengend Snippet: (A) Images of CF488-labeled M13 phages, and Cy3-labeled transferrin in HUVECs after 30 min and 1 h, at 37°C, and 4°C. Quantification of the (B) red fluorescence area (transferrin), and (C) green fluorescence area (M13 phages) in HUVECs following 30 min and 1 h, at 37°C, and 4°C, as shown in panel (A). Data represent mean values obtained from analysis of 5 randomly selected images. b Images of CF488-labeled M13 phages, and Lysotracker Deep Red in HUVECs after 30 min and 1 h. Scale bar, 10 pm.

Article Snippet: An aliquot of 1 µmol CF™ 488A succinimidyl ester (CF488-SE; Biotium) was dissolved in 100 µL anhydrous DMSO to obtain a 10 mM CF488-SE solution.

Techniques: Labeling, Fluorescence