Visualizing Bm16M Intracellular Trafficking

To visualize Bm16M intracellular trafficking, the indicated host cells (5.0 × 10⁴) were seeded on 12-mm coverslips placed on the bottom of wells of 24-well plates and infected with Bm16M-GFP or Bm16M for various lengths of time. To analyze infections of less than 0.5 h, the infected cells were rinsed with 1 × PBS and then fixed with 3.7% formaldehyde at 4°C for overnight before immunofluorescence microscopy analysis (IFMA). Otherwise, at 0.5 h.p.i., the infected cells were washed with 1 × PBS and fresh media supplemented with 40 μg/ml gentamicin was added to kill extracellular bacteria. At the indicated time points post-infection, the infected cells were fixed and IFMA was performed as previously described (Qin et al., 2008 (link), 2011 (link); Pandey et al., 2017 (link)). The primary antibodies used were as follows: goat-anti Brucella, rabbit anti-LAMP-1; rabbit anti-cathepsin D; rabbit anti-mouse LC3; rabbit anti-Calreticulin; rabbit anti-ULK1, rabbit anti-Beclin 1 (Santa Cruz Biotech., Inc, 1:200-500). Samples were stained with Alexa Fluor 488-conjugated and/or Alexa Fluor 594-conjugated secondary antibody (Invitrogen/Molecular Probes, 1:1,000). Acquisition of confocal images, and image processing and analyses were performed as previously described (Qin et al., 2008 (link), 2011 (link); Pandey et al., 2017 (link)).