Flurostar

MSM-NB was inoculated with one single colony (walled cells) or a 1:100 dilution of a 24 h culture (L-form) and incubated at 30^°C overnight. The next morning the overnight culture was diluted in fresh MSM-NB and was grown to exponential phase. Each culture was diluted again in the same medium to an OD 600 nm of 0.02, then aliquoted into 96-well plates. Cultures were grown for 1 h and then challenged with a dilution series of meclizine (made in MSM-NB) to give a final volume of 200 μL. Culture growth was monitored for 12 h at 30^°C, using a FluroStar (BMG LABTECH) plate reader.

CRT released in to 1 ml of culture media from ~ 1 × 10⁵ cells treated with DX and/or TG with or without TUDCA was measured by ELISA [30 (link)]. Briefly, 153 μl of cell-free supernatant was placed into wells of a 96-well ELISA plate with 17 μl of 10 × carbonate buffer, pH 9.6. The protein supernatants were left to bind at 4 °C overnight. The plates were then washed four times with phosphate-buffered saline (PBS) containing 0.1% v/v Tween 20 (PBST). Remaining binding sites were blocked with 5% v/v BSA in PBST at 37 °C for 30 min. Wells were washed a further four times with PBST. Next 100 μl of 1:2000 dilution of rabbit anti-human CRT antibody (Thermofisher PA3-900) diluted in PBST was added to each well for 2 h in 37 °C. The wells were washed again as described above, and a 100 μl of 1:2000 dilution of secondary anti-rabbit HRP-conjugated antibody was added to the wells and the plate incubated 1 h in 37 °C. The reaction was developed for 15 min at RT in the dark and was terminated by adding 50 μl 2N H₂SO₄ to each well. The optical density at 450 nm (OD45nm) of each sample was measured on a BMG Labtech FLUROstar™ plate reader. A CRT standard curve was generated from known concentrations of CRT (Fig. S3).