Glass chamber slides

Bacteria were grown in host cells directly in glass chamber slides (Ibidi, USA) or dried onto a regular glass slide, then fixed with 4 % paraformaldehyde. Cells were permeabilised with 0.5 % triton X at 4 °C for 5 minutes, and then labelled using primary (Table S2) and fluorescently conjugated secondary antibodies. For labelling with the probes HADA and HALA, bacteria were grown in host cells on glass chamber slides, and the probe was added directly to the growth media to a final concentration of 1 mM (a dilution of 1/200). The cells were incubated at 35 °C with 5% CO₂ for 3 hours, then washed with PBS and fixed as above. For labelling with EDA-DA, the probe was added to growing bacteria (3 days post infection) at 1 mM concentration and incubated for 24 hours at 35 °C with 5% CO₂. When labelling was performed in the presence of drugs these were added at the same time as EDA-DA and incubated for 24 hours at 35 °C with 5% CO₂. Cells were fixed and permeabilised as above, and the probe was reacted with azide-derived Alexa fluor 488 using the Click-iT cell reaction buffer kit from Invitrogen (catalog number C10269) following the manufacturers instructions.

iNKT cells were labeled with Fluo-4 (Calbiochem) according to the manufacturer’s instructions, then 7.5× 10⁴ cells were seeded onto glass chamber slides (iBidi) coated with a 1 μg/ml or a 5 μg/ml solution of ICAM-1-Fc and blocked with 2.5% BSA, or coated with poly-L-Lysine. The slides were placed into a 37 ^oC and 5% CO₂ chamber, and images were taken every 20 seconds for 30 minutes using a Nikon Ti-Eclipse inverted wide-field microscope. For analysis of Fluo-4 signal intensity, 25 iNKT cells from each condition were chosen randomly at a single time-point based on bright field images, and the fluorescence intensity of each cell was determined using FIJI/ImageJ2 software (imagej.net/Fiji). For analysis of Fluo-4 signal over time, live cells were manually tracked over 30 minutes (90 frames) with the changes in 2-D mean fluorescence intensity (MFI) gauged on a per cell basis.

HTR-8/SVneo cells were seeded at 2 × 10⁴ cells/well in glass chamber slides (ibidi GmbH, Germany) and incubated for 24 h. Cells were then treated with K+PFBS (0, 0.01, 0.1, 1, 10, 100 μM) for 24 h. Cells were fixed with cold methanol (−20°C) for 10 min and blocked with 1% BSA, 5% normal goat serum and 0.1% tween-20 in PBS for 60 min at room temperature. After blocking, the cells were incubated with primary antibodies overnight at 4°C in humidified chambers. Primary rabbit anti-human Ki-67 antibody (Abcam, Cambridge, MA) was used at a 1:500 dilution. Anti-rabbit IgG antibodies were used as the negative control (R & D system, Minneapolis, MN). Goat anti-rabbit secondary antibodies and Alexa Fluo 594 (Life Technologies, Carlsbad, CA) were used at 1:500. Slides were mounted using mounting medium for fluorescence with DAPI (Vector Laboratories, Burlingame, CA) and examined with a Zeiss Axio Imager widefield fluorescence microscope. Images were taken from each quadrant per well, with an average of two images per quadrant (eight areas examined per treatment per repeat). The ratio of Ki-67 staining was determined by calculating the number of Ki-67 positive cells (red)/DAPI positive cells (blue). Experiments were repeated three times (N=3).