Flexacam c1 camera

Following a virus cross-neutralization test against FAdV-8a and -8b reference strains, as described above, the cells were fixed by adding ice-cold methanol for 5 min. Following serial washing steps and blocking with 3% bovine serum albumin (BSA) for 1 h, in-house-generated polyclonal rabbit anti-FAdV antiserum (diluted 1:500 in PBS) was added to each well overnight. After removal of the rabbit antiserum and washing, the plates were incubated with 1:200-diluted Alexa Fluor 488-conjugated donkey anti-rabbit IgG (Invitrogen, Life Technologies, Carlsbad, CA, USA) in the dark for 1 h. After another wash, cells were stained with 1:1,000 DAPI (4′6-diamidino-2-phenylindole solution; Roche Diagnostics GmbH, Vienna, Austria) for 5 min, subjected to a final wash, and covered with PBS.
Internalization of viral particles was examined and documented with a Zeiss Axiovert 200 M fluorescence microscope (Zeiss, Jena, Germany) coupled to a Flexacam C1 camera and Leica Application Suite X (LAS X) (Leica Microsystems GmbH, Wetzlar, Germany).

RAW 264.7 cells (1 × 10⁴), seeded on black/clear bottom 96-well-microtiter plate, were pre-treated with AJHLE at non-toxic concentrations (12.5, 25, and 50 µg/mL) for 2 h and then exposed or not to LPS (200 ng/mL) for 24 h under standard culture conditions. At the end of the experiment, the fluorescent probe 2′,7′dichlorodihydrofluorescein diacetate (DCFH-DA) (Sigma-Aldrich, Hamburg, Germany), was used to evaluate the intracellular ROS levels [29 (link)]. Precisely, after washing the cells with PBS, fresh culture medium without FBS, and containing 10 µM of DCFH-DA, was added and further incubated under standard culture conditions for 30 min. After this period, the fluorescent probe was replaced with PBS, and the cell fluorescence was measured at 485 nm (excitation) and 535 nm (emission) with a SpectraMax iD3 multi-mode microplate reader (Molecular Devices, USA). Intracellular ROS levels were expressed as percentages of negative control (cells treated with DMSO 0.5% without LPS). Images were captured using a Flexacam C1 camera (Leica Microsystems GmbH, Wetzlar, Germany) connected to a fluorescence microscope (Leica Microsystems GmbH, Wetzlar, Germany) using an excitation/emission filter (480/535 nm) on 40X objective.

BM cells were isolated from the femur and tibia of Balb/c mice and purified by Percoll gradient centrifugation [26 (link)]. Beta-galactosidase activity was determined according to the protocol of Debacq-Chainiaux et al. [72 (link)]. Briefly, the cells were seeded at 24-well plates at a concentration of 2 × 10⁶ cells/mL and were cultured in the presence of 0.01% DMSO (Vehicle) or 1 µM CDDO-Me. After 6 h, the cells were fixed with 0.2% Glutaraldehyde/dH₂O (#354400; Sigma-Aldrich, Munich, Germany) for 10 min, washed twice with PBS, and incubated with the chromogenic buffer containing 150 mM NaCl, 2 mM MgCl₂·6H₂O; 1 mg/mL X-Gal (#203782, 5-Bromo-4-chloro-3-indolyl-β-D-galactopyranoside, Calbiochem, Munich, Germany), and 0.4 mM Nitrotetrazolium Blue Chloride (NBT) (#11585029001, Roche, Munich, Germany) dissolved in phosphate buffer (pH 7.4; 500 mM K₂HPO₄·3H₂O; 500 mM KH₂PO₄) for 18 h. The fixed cells were rinsed with 80% methanol/dH₂O and air dried. The cells positive for beta-galactosidase were stained in blue. Each well was examined using a Leica DMi8 inverted microscope at 10× and 20× magnifications using a Flexacam C1 camera (Leica Microsystems; RSR Ltd., Sofia, Bulgaria). The photos were captured and the cells showing β-galactosidase activity (blue staining) were detected and counted using Image J software.