2.9. Immunofluorescence Staining for LC3B Puncta

To identify autophagosome production through LC3B puncta, cells were immunofluorescence stained. The cells were seeded on coverslips in 6-well plates, pre-treated with astaxanthin for 3 h, and then stimulated with H. pylori for 24 h. The medium was removed, and the cells were washed with PBS and fixed with 4% formaldehyde for 10 min at room temperature. The fixed cells were permeabilized with 0.1% Triton X-100 for 15 min at 15–25 °C. Then, the cells were blocked for 1 h in a blocking solution and incubated for 1 h with primary antibody against LC3B (L7543, Sigma-Aldrich). After washing with PBS, the cells were reacted with rhodamine-conjugated mouse anti-rabbit IgG antibody (sc-2492, Santa Cruz Biotechnology, Dallas, TX, USA) for 1 h. After the removal of the secondary antibody, the cells were washed with PBS and covered with the antifade medium Vectashield containing 4′,6-diamidino-2-phenylindole (DAPI). The preparations were stored for 30 min to allow saturation with DAPI. The cells stained with rhodamine-conjugated antibody were examined under a laser scanning confocal microscope (Zeiss LSM 880, Carl Zeiss Inc., Thornwood, NY, USA) and photographed. Each sample was analyzed using a threshold of >7 dots/cell. LC3B puncta-positive cells were quantified and expressed as % of cells with >7 LC3B puncta/total number of cells.