rabbit anti-human fibronectin pab  (Agilent technologies)

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Non-typeable Haemophilus influenzae major outer membrane protein P5 contributes to bacterial membrane stability, and affects the membrane protein composition crucial for interactions with the human host

doi: 10.3389/fcimb.2023.1085908

Figure Lengend Snippet: Analysis of bacterial pathogenic phenotypes and membrane stability. (A) Bacterial adherence to human type II alveolar epithelial cells (A549) at multiplicity of infection (MOI) of 100 for 30 min. Mean data from three independent experiments (biological replicates) is presented. Bacterial adherence was presented as percentage of CFU recovered per well relative to initial inoculum. (B) Binding of NTHi 3655 wild-type and mutants to human fibronectin. Bacterial (5×10 7 CFU) binding to human fibronectin (0.8-2.0 µg/ml) in 100 µl reactions was analysed by flow cytometry after incubation for 1 hour at 37°C. Rabbit anti-human fibronectin and FITC-conjugated swine anti-rabbit pAbs were used to detect the bacterial-bound fibronectin. Data represent mean values of three independent experiments. (C) Serum killing of NTHi 3655 wild-type and mutants. Bacterial (1.5×10 3 CFU) killing by 5% NHS was analysed by CFU count on chocolate agar. Heat-inactivated serum was included as a negative control and here no bacteria were killed (data not shown). Percentage of bacterial survival was expressed as (T t CFU/T 0 CFU)×100. T 0 represents CFU of sample plated at 0 min; and Tt represents CFU of sample plated at indicated time points. Data represent mean values of three independent experiments. (D) Outer membrane vesicles (OMVs) production among NTHi 3655 wild-type and mutants. OMVs from bacterial cultures were sucrose-density gradient purified and subjected to nanoparticle tracking analysis with a NanoSight NS300. OMV samples were diluted in PBS until 20-120 particles per frame were archived. Settings were optimized using 100nm polystyrene beads, and samples were recorded using the same settings (camera level 12, three recordings of 30 sec each). Recordings were thereafter processed using the NanoSight 3.1 software. Data represents mean values from three independent experiments. (E) Spot viability assay of bacterial survival in response to hyperosmotic environment. Bacteria that were serially diluted (10 9 to 10 4 CFU/ml) was spotted on chocolate agar without sodium chloride (NaCl) (left panel) or supplemented with 50 mM (middle panel) and 100 mM NaCl (right panel). Images were captured using ProtoCOL 3 HD (Synbiosis, UK). The assay was repeated in three independent experiments, and images from a representative experiment were shown. For panel A-D, error bars indicate standard deviations. Differences between wild-type and mutants were calculated by one-way ANOVA for panel (A, D) ; and two-way ANOVA for panel (B, C) *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.005; ****, P ≤ 0.001. WT, NTHi 3655 wild-type; Δ p5 , p5 -knockout mutant (NTHi 3655Δ p5 ); Δ p5 CTD , mutant expressing P5 without CTD (NTHi 3655Δ p5 CTD ); Δ p5::p5, p5- transcomplemented NTHi (NTHi 3655Δ p5::p5 ).

Article Snippet: Bacteria-bound fibronectin was detected with rabbit anti-human fibronectin pAb (Dako, Glostrup, Denmark) and FITC-conjugated swine anti-rabbit pAb (Dako).

Techniques: Infection, Binding Assay, Flow Cytometry, Incubation, Negative Control, Purification, Software, Viability Assay, Knock-Out, Mutagenesis, Expressing

Journal: Matrix biology : journal of the International Society for Matrix Biology

Article Title: Fibroblast-fibronectin patterning and network formation in 3D fibrin matrices.

doi: 10.1016/j.matbio.2017.06.001

Figure Lengend Snippet: Figure 3. Temporal assessment of fibronectin track formation. Cells expressing LifeAct (green) in fibrin matrices were monitored with media containing fluorescent fibronectin (white). A) Initial cell spreading. After one hour of incubation, the matrix was transferred to the microscope for time-lapse imaging. Z-stacks of images were collected at 20 minute intervals. Images show maximum intensity projections. Note that fibronectin is present at the leading edge during cell spreading. Scale bar is 25 m. B) Cell movement within fibronectin network. Cell/fibrin matrix was incubated for 23 hours in PDGF. After one hour of incubation with Rhodamine fibronectin, the matrix was transferred to the microscope for time-lapse imaging. Z- stacks of images were collected at 20 minute intervals. Images show maximum intensity projections. Arrowheads show areas where new fibronectin tracks were created during migration of the cells. Arrows show a cell spreading into a pre-existing fibronectin track. Scale bar is 50 µm.

Article Snippet: For immunostaining, a rabbit anti- human fibronectin polyclonal antibody (sc-9068), a rabbit anti-5 integrin antibody (EPR7854) and a goat anti-human fibronectin polyclonal antibody (sc-6952) were used (Santa Cruz Biotechnology, Inc., Santa Cruz, CA).

Techniques: Expressing, Incubation, Microscopy, Imaging, Migration