Trypan blue

To discriminate between binding and internalization of GFP-BCG, we carried out reduction in green fluorescence of Trypan blue quenched bacteria by excitation energy transfer [26 (link),27 (link)]. To this end, we washed infected or uninfected bone-marrow-derived macrophages in cold phosphate buffered saline (PBS) buffer (137 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄, 2.0 mM KH₂PO₄; pH adjusted to 7.4) and incubated them in PBS on ice for 15 min. Cells were harvested, normalized to a concentration of 1 × 10⁶ cells/50 µL in PBS, and either fixed in 4% paraformaldehyde (PFA; Sigma-Aldrich, Steinheim, Germany) for 10 min at room temperature or processed to quench adherent bacteria. To quench the fluorescence of adherent GFP-BCG, 500 µL Trypan blue (0.4% w/v in PBS; Corning Inc., Corning, NY, USA) was added for 1 min, followed by washing twice in PBS and fixation in 4% PFA for 10 min at room temperature. Quenching with Trypan blue reduced the GFP fluorescence of only adherent bacteria (not internalized bacteria) by excitation energy transfer [26 (link),27 (link)]. Bacterial binding (without Trypan blue treatment) and internalization (with Trypan blue treatment) were analyzed by Attune NxT flow cytometry (Thermo Fisher Scientific, Waltham, MA, USA) and FlowJo software v10 (FlowJo LLC, Ashland, OR, USA).

Further, trypan blue stain assay was conducted for the determination of a viable cell count in order to confirm the cytotoxic effects of experimental conditions. Neuro-2a cells were cultured in a 6-well plate (2 × 10⁵ cells/well) under C, N or B conditions for 8, 24 or 48 h. After incubation, cells were detached by trypsinization. The cell pellet was collected and re-suspended in PBS solution followed by staining with trypan blue (CORNING, Christiansburg, VA, USA). The viable cells, which did not take up the stain, were counted with a hemocytometer under a microscope (Olympus, Tokyo, Japan).

Transiently transfected INS-1 cells were harvested at 48 h post-transfection by using a cell scraper to dislodge all cells, and 10 μL of cells were collected from each well. Cell viability was determined using trypan blue (Corning, Corning, NY, USA) staining using the TC-10 Automated Cell Counter (BioRad, Hercules, CA, USA). Comparisons were made by paired t-test, including all technical and biological replicates; statistical significance was determined by p < 0.05.