Ti e inverted microscope

To visualize killing by cephalexin, a 20-h overnight culture was diluted 1:5000 and incubated for 90 min. The resulting exponential phase culture was washed with MgSO₄ and 2 µl of cells was seeded onto an MHB-agarose pad (2% w/v) containing cephalexin (50 µg/ml). Cells were incubated at 37 °C and killing was monitored for 6 h. Images were obtained using a Nikon Ti-E inverted microscope with a 60x objective.
To visualize persisters, cells were isolated as described above. The resulting sample was resuspended in 10 µl MgSO₄ and 2 µl of cells was seeded onto an MHB-agarose pad (2% w/v) with or without cephalexin (50 µg/ml). Cells were incubated at 37 °C and growth was monitored for 12 h. Images were obtained using a Nikon Ti-E inverted microscope with a 60x objective.

Steady fluorescent imaging data for Fig. 1a, Supplementary Figure 7b and 8 were acquired by inverted DeltaVision Elite widefield fluorescence microscope (GE Healthcare Life Sciences). Bacterial samples fixed with 2% PFA were spotted on 1.0% PBS agarose pad. DNA staining (Supplementary Figure 7b) was carried out by incubating fixed cells with 1 μg ml-1 Hoechst 33342 (Sigma) at room temperature for 10 min, and washed with PBS + 0.05% Tween-80 for three times to minimize dye carried-over. Steady fluorescent imaging data for Supplementary Figure 1 was acquired on a Nikon TI-E inverted microscope. Time lapse imaging was performed on a Nikon TI-E inverted microscope with an environmental chamber maintained at 37 °C. Mid-log-phase cultures were injected into a B04A microfluidic bacteria plate (CellASIC) to optimal density, then the cell chamber perfused with 7H9 medium. After 8 h, 7H9 medium supplemented with 10, 20, or 30 μg ml⁻¹ rifampicin was perfused into parallel flow chambers within the same chip and continued for 30 h.

To test the angiogenic potential of mast cells in the presence of healthy and degenerate IVD cells, media from the DCCM-Mast cell cultures was evaluated for blood vessel formation using the tubular assay. Human Umbilical Vein Endothelial Cells (HUVECs) were expanded in Medium 200 with Low Serum Growth Supplement (LSGS) (ThermoFisher) and seeded in 96 well plates at 4.0 × 10⁴ cell/well on wells pre-coated with 32 μl of Geltrex (ThermoFisher – A1413201)^{64 (link)}. Conditions included (1) Standard HUVEC media with LSGS (Positive control), (2) HUVEC media without LSGS (negative control), (3) media from mast cells in basal conditions, and (4) the healthy and (5) degenerate DCCM-mast cell groups (N = 4). Media from each group was filtered using PALL Microsep Advance Centrifugal Devices (PALL Corporation) to retain the soluble factors greater than 3,000 Da and resuspended in HUVEC media (Medium 200) without LSGS before being applied to HUVECs. HUVECs were incubated in conditions described above at 5%CO₂ at 37 °C for 6 hours. After incubation, 4 mM Calcein AM was added and images captured at ×4 magnification using TiE Nikon Inverted Microscope. The percent (%)area of tubular fluorescence was quantified using ImageJ⁸⁰ .