8 well chambers

Dilutions of B. pseudomallei bacteria were prepared in Dulbecco’s phosphate buffered saline (DPBS). OECs and TgSCs were seeded at 5000 cells/well in 8-well chambers (Sarstedt), in glial medium. After 12 h, bacteria (MOI 75:1) and/or cell debris (final concentration: 33 μg/ml) were added and incubated with cells for 24 h. Cells were then rinsed in 1x HBSS and were fixed for 20 min in 4% paraformaldehyde (PFA) in DPBS. Cells were washed and incubated in blocking buffer for 1 h before immunolabelling for B. pseudomallei as described above. Nuclei were stained with DAPI. Images of the cells were taken with a confocal microscope (Olympus FV1000); the number of MNGCs were counted (n = 5 fields of view with 50–70 cells in each). For the assay which investigated cellular responses to B. pseudomallei lacking the BimA protein, OECs and TgSCs were incubated with MSHR520Δ_CapΔ_BimA (MOI 75:1) for 48 h.

Cell microscopy studies were performed in 8-well chambers (Sarstedt, Germany). 1.5*10⁴ cells were seeded 24 hours before the experiment in 300 μl of complete medium. Alexa Fluor 555 siRNA-labeled complexes were incubated at 37°C for 4 hours. The cells were washed 3 times with Phosphate Buffered Saline PBS (GIBCO, Germany) and then were fixed in 4% formaldehyde solution and stained with DAPI using Vectashield Mounting Medium (Vector Laboratories, Burlingame). Images were acquired in multichannel acquisition mode with Axio Observer Z1 (Zeiss), with a 20 X objective and the images were processed using Axio vision software.