Journal: Frontiers in Immunology

Article Title: Development and characterization of segment-specific enteroids from the pig small intestine in Matrigel and transwell inserts: insights into susceptibility to porcine epidemic diarrhea Virus

doi: 10.3389/fimmu.2024.1451154

Figure Lengend Snippet: Porcine enteroids on transwell culture (PETCs) derived from three regions (duodenum, jejunum, and ileum) of porcine small intestine inoculated with PEDV and mock. Representative images of PETCs inoculated with porcine epidemic diarrhea virus (PEDV, USA/Colorado/2013) at 1.58 x 10 5 TCID50/mL or mock-inoculated and incubated for 24 h (n=4); Bright-field images of PEDV-inoculated cells showing cytopathic changes such as increased detachment of cells (A–C) as compared to the mock-inoculated (D–F) . Immunofluorescence images showing for PEDV nucleocapsid (N) protein (green) following staining with an IgG1 fluorescein isothiocyanate (FITC)-conjugated anti-PEDV N protein monoclonal antibody (Medgene) at 1:100 dilution in PBS pH 7.4 with 0.1% bovine serum albumin (BSA; Jackson Immuno Research). Intense green fluorescent signals can be observed in the infected PETCs (G–I) as compared to the mock-inoculated (J–L) . (M) Bar graph showing the trend of integrated fluorescence intensity values (y-axis) obtained from Image J analysis of 200X magnified images representative of two experiments. The product of the mean fluorescence intensity and the percentage of area with fluorescent signal was termed integrated fluorescence intensity. (N) Bar graph representing normalized gene expression of PEDV N-gene against EIF3K endogenous control gene in PETCs derived from different small intestinal segments. The relative quantification values (y-axis) data obtained from ΔΔCt analysis showed an average of four replicates (Error bars = SEM) with P value < 0.001 (***) denoting statistical significance, while ns denotes no significance.

Article Snippet: After washing twice each well with 200 μL PBS, 100 μL of fluorescein isothiocyanate (FITC)-conjugated mouse anti-PEDV nucleocapsid (N) protein IgG1 monoclonal antibody (Medgene, Brookings, SD, USA) diluted 1:100 in PBS pH 7.4 containing 0.1% bovine serum albumin (BSA; Jackson Immuno Research, West Grove, PA, USA) was added per well and incubated for 1 h at 37°C.

Techniques: Derivative Assay, Virus, Incubation, Immunofluorescence, Staining, Infection, Fluorescence, Gene Expression, Control, Quantitative Proteomics