collagen coated transwell filter inserts  (Genesee Scientific)

Journal: Frontiers in Microbiology

Article Title: Cronobacter sakazakii ATCC 29544 Translocated Human Brain Microvascular Endothelial Cells via Endocytosis, Apoptosis Induction, and Disruption of Tight Junction

doi: 10.3389/fmicb.2021.675020

Figure Lengend Snippet: Adhesion, invasion, intracellular replication, and translocation of C. sakazakii in HBMEC. (A) The number of C. sakazakii cells that adhere to HBMEC. (B) The number of C. sakazakii cells in HBMEC after 2 h invasion and intracellular bacteria counts at 2, 4, and 6 h post-invasion. (C) HBMEC were incubated in transwell inserts. (D) The counts of C. sakazakii that translocated HBMEC monolayers were calculated. The strain ATCC 29544 performed the strongest invasion and translocation abilities compared to other strains ( P < 0.01). Bars represent the means ± standard deviations ( n = 3). Mean values with different lower-case letters are statistically different ( P < 0.05).

Article Snippet: The transwell systems were employed for transcytosis assay as previously described , HBMEC were seeded (2 × 10 4 cells/insert) onto the inner surface of collagen-coated transwell inserts (24 well format, 3.0-μm pore size polycarbonate filter; Corning, NY, United States) which were placed in wells of a 24-well plate ( ).

Techniques: Translocation Assay, Bacteria, Incubation

Journal: Burns & Trauma

Article Title: Molecular mechanism mediating enteric bacterial translocation after severe burn: the role of cystic fibrosis transmembrane conductance regulator

doi: 10.1093/burnst/tkaa042

Figure Lengend Snippet: Decrease of CFTR, ZO-1, occludin and E-cadherin in intestinal epithelia post severe burn. (a) Bacterial counts of homogenates of lymph nodes, spleen and liver of sham or burnt mice. Data are means ± SEM from at least 3 independent experiments, n = 4. (b) Western blotting of CFTR, occludin, E-cadherin and β-actin of the ileum of C57BL/6 mice with (burn) or without (sham) 30% TBSA full-thickness cutaneous burn (left panel) and statistical analysis (right panel). Band B (160KD) and Band C (180KD) of CFTR showing different levels of glycosylation of CFTR are indicated by arrows. As band C (180KD) represents the fully glycosylated mature CFTR protein, which functions as an ion channel, the quantitative bar graph corresponds to band C. (c) Representative images and statistical analysis of immunofluorescence staining for CFTR and ZO-1 in the ilea of sham and burnt mice. Scale bar = 20 μm. To prepare the monolayer, Caco-2 cells were seeded in collagen-precoated permeable polycarbonate membrane transwell with 0.4-μm pores and used for experiments after confluence. Caco-2 monolayers were exposed to 1% O 2 for 6 or 12 hours as hypoxia treatment or without hypoxia treatment (H, hypoxia). Real-time quantitative PCR (d) and western blot (e) analyses of CFTR, ZO-1, occludin and E-cadherin were performed. (f) TER measurement of Caco-2 monolayers with or without hypoxia. All TER values were normalized to the control group (normoxia). One-way ANOVA followed by Tukey’s post hoc test was used when there were more than 2 groups. The experiments were repeated at least twice. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. CFTR cystic fibrosis transmembrane conductance regulator, DAPI 4′,6-Diamidino-2-phenylindole, MLN mesenteric lymph nodes, N normoxia, OCLN occludin, TER transepithelial resistance ZO -1 zonula occludens-1, TBSA total body surface area

Article Snippet: Caco-2 cells were cultured on collagen-coated transwell polycarbonate membrane filter inserts (Corning).

Techniques: Western Blot, Immunofluorescence, Staining, Real-time Polymerase Chain Reaction