Synergy ht fluorescent

Human VLDL (BTI Biomedical Technologies) was labeled with 50 μg of FITC dissolved in DMSO (1 mg/ml) (Sigma-Aldrich) for 2 h at room temperature. Next, conjugated VLDL was separated from free fluorochrome by gel filtration using PD10 column (GE Healthcare). We injected 100 μg of FITC-VLDL to the tail vein of ApoE- and double KO mice. Blood samples were collected into EDTA-coated tubes 1 and 15 min after injection. Plasma samples were diluted 1:20 in PBS and fluorescence was read using a BioTek synergy HT fluorescent plate reader. For each mouse, data were normalized to fluorescent intensity measured at 1 min.

Mutant or wild-type strains, with the pGLOW reporter, were grown in EVCC + Aviguard. Oxyrase (Sigma-Aldrich; St. Louis, MO) was added to the medium (1:20 final dilution) and overlaid with mineral oil to create low oxygen conditions. Salmonella starting cell density was 10⁵ CFU/mL. The lyophilized Aviguard was reconstituted as recommended by the manufacturer (Lallemand Animal Nutrition) in sterile saline with Oxyrase and used to inoculate EVCC cecal medium (1:20 dilution) (7.99 Log₁₀ viable cells/mL). The viability of the rehydrated Aviguard was determined microscopically using LIVE/DEAD BacLight stain (Molecular Probes). Sterile 96-well, black-walled polystyrene microtiter plates with a clear bottom (Fisher Scientific) were used to monitor Salmonella fluorescence with a BioTek Synergy HT 96-well fluorescence microtiter plate reader at 37°C. A filter was added to the BioTek Synergy HT fluorescent plate reader (BioTek; Winooski, VT) to record pGLOW fluorescence based on its excitation frequency (Drepper et al., 2007 (link)). Fluorescence was recorded every 30 min. Strains were run in triplicate.