Black flat 96 well plates

Cytotoxicity was evaluated using a propidium iodide assay. Briefly, human lung adenocarcinomic A549 cells were plated on black flat 96-well plates (Greiner) at a concentration of 0.5 × 10⁶ cells/mL in DMEM GlutaMAX (Gibco) supplemented with 10% fetal bovine serum for 24 h in a humidified atmosphere at 37 °C and 5% CO₂. The medium was then removed and fresh medium with 2.5 µg/mL of propidium iodide and tested compounds (128 µg/mL of II.b and 0.2 µg/mL for recombinant Hla) was added. Absorbance at 618 nm was measured every 10 min over 7 h. Each concentration was made in triplicate and mean values are displayed with standard deviation.

The 1 × 10⁴ cells were seeded in black flat 96-well plates (Greiner Bio-One, Kremsmünster, Austria). For measurement of intracellular calcium concentration Cal-520 No Wash Calcium Assay (Abcam, Cambridge, UK) was used. Five µM of the fluorescent dye Cal-520 (Abcam, Cambridge, UK) were added to Hank’s Balanced salt solution (HBSS, Gibco, Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 20 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer (LONZA, Basel, Switzerland), 0.02% Pluronic^TM F-127 (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA) and 1 mM Probenecid (Sigma-Aldrich St. Louis, MO, USA) from which 100 µL were given to each well. Afterward, incubating the plate for 90 min at 33 °C or 39 °C and adding 100 µL Probenecid dissolved in Minimum Essential Medium (MEM, Gibco, Thermo Fisher Scientific, Waltham, MA, USA) intracellular calcium concentration was measured at Ex/Em = 490/525 nm with Tecan infinite F2000 Pro (Tecan, Männedorf, Switzerland).

Cytotoxicity was evaluated using a propidium iodide assay. Briefly, human lung adenocarcinomic A549 cells were plated on black flat 96-well plates (Greiner, Pleidelsheim, Germany) at a concentration of 0.5 × 10⁶ cells/mL in DMEM GlutaMAX (Gibco, Waltham, MA, USA) supplemented with 10% fetal bovine serum for 24 h in a humidified atmosphere at 37 °C and 5% CO₂. The medium was then removed, and fresh medium with 2.5 µg/mL of propidium iodide and tested compounds (64 µg/mL of II.c and 0.2 µg/mL for recombinant Hla) was added. Plates were incubated for 7 h at 37 °C, and the cytotoxicity induced was evaluated by measuring propidium iodide (PI) incorporation into cells exposed to toxin or compounds using a fluorescence cell sorter (Spark R, TECAN, Zürich, Switzerland). Each concentration was made as a triplicate, and mean values are displayed with standard deviation.