Resorufin

To asses cell viability in organoids treated with LM or LM-BOM, on the 20th day of culture, the media were changed with fresh BOM, and then resazurin (Merck, Germany) was added to the medium to the final concentration of 10 μg/mL. The plates were incubated for 6 h. Resorufin fluorescence (excitation at 560 nm, emission at 590 nm) was measured using Synergy H4 Hybrid multimode microplate reader (BioTek, United States) in technical triplicates. As a positive control of dying cells, organoids in LM-BOM conditions were treated from day 16 with 40 μM taxol (Sigma, United States) or killed on day 20 by treatment with 70% ethanol for 5 min.

The antimicrobial activity of the different compounds was assessed by broth microdilution, following the Clinical and Laboratory Standards Institute (CLSI) guidelines [41 ] and as described previously [40 (link)], using stock solutions of the compounds in DMSO, due to the negligible solubility of Clf in water [37 (link)]. Briefly, M. avium was grown in Middlebrook 7H9 medium to the exponential phase and 1 to 5 × 10⁵ CFU/mL of bacteria was seeded in 96-well plates with increasing concentrations of the compounds. Each condition was tested in triplicate. The plates were incubated at 37 °C in a humid atmosphere. After 6 days of incubation, bacterial viability was assessed by resazurin reduction. Then, 10% (v/v) of resazurin (Sigma-Aldrich, St. Louis, MO, USA) at 2.5 mM in phosphate-buffered saline (PBS, Sigma-Aldrich, St. Louis, MO, USA) was added to each well, and after 24 h of incubation at 37 °C, the fluorescence of resorufin, resulting from the conversion of resazurin by metabolically active cells, was measured at λ_ex = 530 nm and λ_em = 590 nm in a Synergy^TM Mx microplate reader from BioTek (Agilent Technologies Inc., Santa Clara, CA, USA) using the Gen5 software also from BioTek. The results are expressed as the percentage of the fluorescence obtained in experimental wells relative to the fluorescence obtained in nontreated wells.

Cannabinoids are known to bind serum proteins; therefore, drug sensitivity was assessed in low serum conditions (1.5% FBS). Cells were plated (1500/well) into tissue culture treated 384-well plates (Corning, New York, NY, USA) using a Multidrop Combi (Thermo Scientific, Waltham, MA, USA). Medulloblastoma and ependymoma cells were incubated for 1 and 24 h, respectively, prior to the addition of drug. Drugs (either single drugs or drug combinations) were dispensed using an HP300 digital dispenser (Tecan, Mannedorf, Switzerland) with concentrations indicated in the text. Cells were treated for 72 h and incubated with alamar blue (2.5% methylene blue, 1 mM potassium hexacyanoferrate (III), 1 mM potassium hexacyanoferrate (II) trihydrate, and 0.6 mM resazurin (all from Sigma-Aldrich)) for the final 6 h of treatment. Resorufin fluorescence was detected using a SynergyMX plate reader (Biotek, Winooksi, VT, USA) with 570 nm excitation and 590 nm emission. Data were expressed as a percentage of DMSO-treated controls present on each plate. The ED50 was interpolated from a best-fit dose–response curve determined using Prism v8 (GraphPad Software, San Diego, CA, USA). Drug interactions were analyzed using Combenefit software (Cambridge University, Cambridge, UK) [45 (link)].