Journal: The FASEB Journal

Article Title: Versatile human in vitro triple coculture model coincubated with adhered gut microbes reproducibly mimics pro‐inflammatory host‐microbe interactions in the colon

doi: 10.1096/fj.202101135r

Figure Lengend Snippet: FIGURE 1 A triple coculture model consisting of a collagen coating with confluent THP-1, LS-174T and T84 cells in DMEM/F-12 at the basolateral side, in indirect contact with apical microbes adhered to a solidified agar-mucin layer covering the Transwell polycarbonate membrane filter (0.4 µm pores) was developed to study host-microbe interactions in the human gut

Article Snippet: The T84 cell line (CLS 300354) and LS- 174T cell line (CLS 300392) were obtained from Cell Lines Service (Eppelheim, Germany).

Techniques: Membrane

Journal: The FASEB Journal

Article Title: Versatile human in vitro triple coculture model coincubated with adhered gut microbes reproducibly mimics pro‐inflammatory host‐microbe interactions in the colon

doi: 10.1096/fj.202101135r

Figure Lengend Snippet: FIGURE 2 Triple cocultures morphologically organize in networks producing thicker and denser MUC2 layers than T84 monocultures. (A) F-actin, nuclei, MUC2 mucin staining and transmission image showing morphological characteristics of T84 monolayer (top) and triple coculture (bottom) cell models as visualized by confocal microscopy. Blue arrows: epithelial T84 cells; White arrows: LS-174T cells; Yellow arrows: Macrophage-like THP-1 cells; Green arrows: apoptotic structures. (B) The thickness of MUC2 layers in monolayers versus triple cocultures prior to microbial exposure (n = 6). (C) Example of a cross section and top view of the MUC2 layer formed on top of the monolayer and triple coculture host cells. *p = .0081

Article Snippet: The T84 cell line (CLS 300354) and LS- 174T cell line (CLS 300392) were obtained from Cell Lines Service (Eppelheim, Germany).

Techniques: Staining, Transmission Assay, Confocal Microscopy

Journal: International journal of molecular sciences

Article Title: shRNA-mediated XRCC2 gene knockdown efficiently sensitizes colon tumor cells to X-ray irradiation in vitro and in vivo.

doi: 10.3390/ijms15022157

Figure Lengend Snippet: Figure 3. Knockdown of XRCC2 by shRNA inhibited cell growth of T84 cells. Cells were transfected with either shRNA-XRCC2 or shRNA-SC. The effect of XRCC2 suppression on cell growth in T84 cell line was examined by MTT assay. The values are presented as the mean ± SD (n = 12). * p < 0.05, ** p < 0.01 compared with the control group.

Article Snippet: T84 cells were stably transfected with either shRNA XRCC2 plasmid (sc-36861-SH, Santa Cruz, Dallas, TX, USA) (shRNA-XRCC2) or control shRNA plasmid-A (sc-108060, Santa Cruz) as scramble shRNA (shRNA-SC), using Lipofectin reagent (Invitrogen, Carlsbad, CA, USA), according to the manufacturer’s instruction.

Techniques: Knockdown, shRNA, Transfection, MTT Assay, Control

Journal: Journal of nuclear medicine : official publication, Society of Nuclear Medicine

Article Title: 124 I-Labeled Engineered Anti-CEA Minibodies and Diabodies Allow High-Contrast, Antigen-Specific Small-Animal PET Imaging of Xenografts in Athymic Mice

doi:

Figure Lengend Snippet: Schematic drawing shows domain structure of parental intact T84.66 antibody (A), T84.66 diabody (B), and T84.66 minibody (C). Anti-CEA minibody (80 kDa) and diabody (55 kDa) were derived from T84.66, a high-affinity and highly specific antibody that recognizes an epitope on A3 domain of CEA. Minibody (C) has glycine-serine–rich 18-amino-acid linker (GS18) between variable light (VL) and variable heavy chains (VH) and human IgG1 hinge (H) joining them to CH3 domain, also derived from human IgG1. Gene encoding diabody (B) encodes glycine-serine–rich 8-amino-acid linker (GS8) between VL and VH regions. Amino acid sequence is GSTSGGGSGGGSGGGGSS for GS18 and GGGSGGGG for GS8.

Article Snippet: Briefly, the T84.66/ GS18 minibody and diabody were expressed in NS0 murine myeloma cells using the pEE12 expression vector and a cytomegalo-virus promoter and glutamine synthetase as the selectable marker (Lonza Biologics) ( 17 ).

Techniques: Derivative Assay, Sequencing

Journal: Journal of nuclear medicine : official publication, Society of Nuclear Medicine

Article Title: 124 I-Labeled Engineered Anti-CEA Minibodies and Diabodies Allow High-Contrast, Antigen-Specific Small-Animal PET Imaging of Xenografts in Athymic Mice

doi:

Figure Lengend Snippet: Size-exclusion HPLC analysis of 124I-radiolabeled anti-CEA minibody (A) and diabody (B). 124I was conjugated to T84.66 minibody and T84.66 diabody as described in Materials and Methods. Radiolabeling efficiency was determined by integrating areas on HPLC trace and determining radioactivity associated with 80-kDa protein peak for minibody or 55-kDa peak for diabody as percentage of total radioactivity eluted. Labeling efficiencies were 33% (not shown) or 46% for minibody (A) and 88% for diabody (B). Peak fractions (based on protein absorbance) were pooled for animal studies. Smaller peaks represent unincorporated label and low-molecular-weight components.

Article Snippet: Briefly, the T84.66/ GS18 minibody and diabody were expressed in NS0 murine myeloma cells using the pEE12 expression vector and a cytomegalo-virus promoter and glutamine synthetase as the selectable marker (Lonza Biologics) ( 17 ).

Techniques: Radioactivity, Labeling, Molecular Weight

Journal: PLoS ONE

Article Title: Aeromonas sobria serine protease decreases epithelial barrier function in T84 cells and accelerates bacterial translocation across the T84 monolayer in vitro

doi: 10.1371/journal.pone.0221344

Figure Lengend Snippet: A: T84 cells were cultured in a Transwell system and then infected with several types of A . sobria strains. After 3 hr of infection (MOI = 5), the reduction in TER was measured. The TER value at 0 hr of infection was taken as 100%. NT: The TER value was measured without bacterial infection. The experiments were performed in triplicate. The data are mean ± SD (error bars). *p<0.01. B: The ability of the A . sobria strains to translocate across the intestinal epithelial cells (T84 cells) at 6 hr after infection (MOI = 5) was assessed using quantitative cultures of medium obtained from the lower chambers. NT: The experiment was done without bacterial infection. ND: The bacterial translocation could not be detected in this experimental condition. The experiments were performed in triplicate. The data are mean ± SD (error bars). C: The proteolytic activity in the culture supernatant of each A . sobria strain was measured as described in the text. The experiments were performed in triplicate. The data are mean ± SD (error bars). D: The presence of ASP in the culture supernatant of each strain was immunologically detected by a western blotting analysis as described in the text.

Article Snippet: The intestinal epithelial cell line T84 was obtained from the European Collection of Authenticated Cell Cultures (ECACC).

Techniques: Cell Culture, Infection, Translocation Assay, Activity Assay, Western Blot

Journal: PLoS ONE

Article Title: Aeromonas sobria serine protease decreases epithelial barrier function in T84 cells and accelerates bacterial translocation across the T84 monolayer in vitro

doi: 10.1371/journal.pone.0221344

Figure Lengend Snippet: A: The asp gene of A . sobria 288 strain was knocked out. The immunological analysis using western blotting revealed that the asp -knocked-out strain (#288 ΔASP) did not produce ASP and the complemented strain (#288 ΔASP::ASP) produced ASP again. B: The proteolytic activity in the culture supernatant of each strain. The experiments were performed in triplicate. The data are mean ± SD (error bars). C: T84 cells were cultured in a Transwell system and then infected with the wild-type A . sobria strain (#288), the asp -knocked-out strain (#288 ΔASP), or the complemented strain (#288 ΔASP::ASP). After 6 hr of infection (MOI = 5), the ability of these A . sobria strains to translocate across the T84 cell monolayer was assessed in the same way as that described in the legend. The experiments were performed in triplicate. The data are mean ± SD (error bars). *p<0.01, **p<0.05.

Article Snippet: The intestinal epithelial cell line T84 was obtained from the European Collection of Authenticated Cell Cultures (ECACC).

Techniques: Western Blot, Produced, Activity Assay, Cell Culture, Infection

Journal: PLoS ONE

Article Title: Aeromonas sobria serine protease decreases epithelial barrier function in T84 cells and accelerates bacterial translocation across the T84 monolayer in vitro

doi: 10.1371/journal.pone.0221344

Figure Lengend Snippet: A: T84 cells were cultured in a Transwell system, and the TER value was measured in the presence of various concentrations (nM) of ASP or absence (NT) of ASP. We also examined the effect of the serine protease inhibitor PMSF on the action of ASP. B: The passive diffusion of FITC-labeled dextran molecules across the T84 monolayer (from the apical side to the basolateral side) treated with ASP was measured. All experiments were performed in triplicate. The data are mean ± SD (error bars).

Article Snippet: The intestinal epithelial cell line T84 was obtained from the European Collection of Authenticated Cell Cultures (ECACC).

Techniques: Cell Culture, Protease Inhibitor, Diffusion-based Assay, Labeling

Journal: PLoS ONE

Article Title: Aeromonas sobria serine protease decreases epithelial barrier function in T84 cells and accelerates bacterial translocation across the T84 monolayer in vitro

doi: 10.1371/journal.pone.0221344

Figure Lengend Snippet: T84 cells were treated with (nM) or without (NT) various concentrations of ASP before the extraction. After the extraction, we detected the proteins constituting the junctional complexes by using a specific antibody against each protein shown in the figure. The results of quantitative analysis of the amount of blotted protein are also shown below the image of western blotting. These experiments were performed in triplicate. The data are mean ± SD (error bars).

Article Snippet: The intestinal epithelial cell line T84 was obtained from the European Collection of Authenticated Cell Cultures (ECACC).

Techniques: Extraction, Western Blot

Journal: PLoS ONE

Article Title: Aeromonas sobria serine protease decreases epithelial barrier function in T84 cells and accelerates bacterial translocation across the T84 monolayer in vitro

doi: 10.1371/journal.pone.0221344

Figure Lengend Snippet: T84 cells were treated with or without (NT) 500 nM ASP for 6 hr. The cells were reacted with a specific antibody against nectin-1, nectin-2, and E-cadherin and visualized using the secondary antibody conjugated with a fluorescent substance, Cy5 or FITC. Nuclei were stained with PI. The merged images are shown in each panel. Merge 1: nectin-2 and E-cadherin, Merge 2: afadin and E-cadherin, and Merge 3: nectin-1 and E-cadherin. Z: Z-stack showing entire sample volume image was also shown.

Article Snippet: The intestinal epithelial cell line T84 was obtained from the European Collection of Authenticated Cell Cultures (ECACC).

Techniques: Staining

Journal: PLoS ONE

Article Title: Aeromonas sobria serine protease decreases epithelial barrier function in T84 cells and accelerates bacterial translocation across the T84 monolayer in vitro

doi: 10.1371/journal.pone.0221344

Figure Lengend Snippet: A: T84 cells were infected (MOI = 1) with or without (NT) A . sobria strain 120, 123 or 288. Bacterial internalization was confirmed using the Aeromonas -specific probe FITC-AER66 as described in Materials and Methods ( fluorescence image ). The figure shown as ‘Merge’ is a superposition of the fluorescence image ( left side ) and the blight image ( center ). B: T84 cells were infected (MOI = 1) with A . sobria strain 120, 123 or 288. The number of bacteria survived from gentamicin protection assay was determined and indicated as colony forming unit (CFU). The experiments were performed in triplicate and the data are means ± SD (error bars).

Article Snippet: The intestinal epithelial cell line T84 was obtained from the European Collection of Authenticated Cell Cultures (ECACC).

Techniques: Infection, Fluorescence, Bacteria