96 well flat bottom microtiter plate

The number of parasites in popliteal lymph nodes was quantified by limiting dilution assay, five weeks post-treatment termination. Briefly, popliteal lymph nodes were weighed before homogenization and then serially diluted in 96-well flat bottom microtiter plates (Sarstedt, Numbrecht, Germany) with Schneider’s insect medium (Sigma, St. Louis, MO, USA) supplemented with 20% fetal bovine serum (FBS) (Gibco, Paisley, UK). The number of viable parasites per mg of tissue was determined from the highest dilution at which promastigotes could be grown after 7 days of incubation at 26 °C [28 (link)].

Inhibition of biofilm formation was evaluated against the P. aeruginosa PAO1, PA03, PA05, PA07, PA08, PA09, PA10, PA14, PA21, PA22, PA31, PA35, and RP73 strains using the MTT assay as previously described by Mardirossian and colleagues [17 (link)]. Biofilm eradication assays were performed against P. aeruginosa strains PAO1, PA08, PA09, and RP73, as previously reported by Bonaventura et al. [18 (link)]. After an 18 h incubation in MH broth in 96-well flat-bottom microtiter plates (Sarstedt, Milan, Italy), the bacterial biofilms were incubated 24 h at 37 °C with different concentrations of D-BMAP18 in 50 μL of MH broth. Before peptide addition, some samples were pre-incubated for 1h at 37 °C in the absence or presence of 128 μg/mL of bovine DNase I (Sigma-Aldrich), or 10 mg/mL of N-Acetyl-L-cysteine (Sigma-Aldrich) or 1 mg/mL of Alginate lyase (Sigma-Aldrich). After incubation, the MTT assay was performed as reported [17 (link)].

Compound potency was assessed by dose-response assays in 96-well, flat-bottom microtiter plates (Sarstedt) or in 384-well, flat-bottom microtiter plates (Corning) as previously described (67 (link)). Plates were incubated at 30°C for the indicated time period. Growth was quantified by measuring OD₆₀₀, and the results were corrected for background media. All strains were assessed in biological-duplicate experiments with technical duplicates. Growth was normalized to the levels seen with the untreated controls, and data were plotted as a heat map using Java TreeView 1.1.6r4.
Dose-response matrixes (checkerboard assays) were performed in a similar manner except that the titers of 2-fold serial dilutions of compound 1 (indicated along the x axis of the checkerboard) and compound 2 (indicated along the y axis) were determined. Values representing the fractional inhibitory concentration index at 90% growth inhibition (FICI₉₀) were calculated as previously described (68 (link)).
BU-CMD hit compounds were newly supplied and dissolved in DMSO, 4EGI-1 (Tocris Biosciences) and rapamycin (BioShop) were dissolved in DMSO, fluconazole (Sequoia Research Products) was dissolved in sterile double-distilled water (ddH₂O), and calcium chloride (BioShop) was used to supplement yeast nitrogen base (YNB) media, as indicated.

The cytotoxic effect of tested compounds and their combinations (1:1 ratio) against bladder epithelial cells was evaluated using MTT assay. For this purpose, T24 (ATCC® HTB­4™) cells were seeded into each well of a 96-well flat-bottom microtiter plate (Sarstedt, Newton, NC, USA) to adhere overnight. After 24 h from seeding, cells were treated with LL-37, doxycycline, CSA-13 and CSA-131 (2, 4, 10, 20 and 50 μM) and combinations of LL-37 with doxycycline, LL-37 with CSA-13 and LL-37 with CSA-131 (ranging from 2 μM to 50 μM; in 1:1 ratio). After 1 h incubation of cells with indicated therapeutic agents (37 °C with 5% CO₂), the MTT salt working solution (100 μL/well at the final concentration of 0.5 mg/mL) was added to each well and incubated for another 2–4 h. Working medium was removed and DMSO was added (100 μL/well) to solubilize formazan crystals. Absorbance was measured at 550 nm using Varioskan LUX. The percentage of cell viability was calculated as (absorbance of treated cells/absorbance of untreated cells) × 100%.