Glomax discover

For the evaluation of the Reactive Oxygen Species (ROS) produced and released in vitro, cells were seeded as described before for the MTT test; the analysis were performed 24, 48, 72 and 96 hours after the addition of clusters in culture medium; a ROS-Glo™ H₂O₂ Assay (Promega) was used, following the producer protocol. Unstained cells are reported as negative control. The Non-Lytic assay was applied and the relative luminescence units were measured by a plate reader (GloMax Discover, Promega). For solution studies, triplicate samples of metallic clusters in water (7 μM) were tested using ROS-Glo™ H₂O₂ Assay (Promega), following the manufacturers’ protocol. An opaque white 96 wells plate was loaded with 50 μl of each sample dispersed in H₂O₂ (5 mM) saturated water, followed by the incubation with H₂O₂ substrate solution and ROS-Glo detection solution. After 20 min incubation at room temperature, the luminescence was measure by GloMax Discover (Promega) plate reader. The luminescence was recorded continuously every 15 minutes for 5 hours.

3T3-L1 pre-adipocytes were seeded and grown on 96-well plates until visual confluency was reached by microscopic inspection. Cells were exposed to different TPM concentrations for 24 h. To quantify the cell viability, after removing supernatants and rinsing the cells with warm PBS, 200 µL of the 0.5 mg/mL MTT labelling reagent was added into each well and incubated at 37 °C and 5% CO₂ covered by aluminum foil for 2.5–3 h. A total of 100 µL of DMSO was added to each well after emptying the plate. When the crystals were solubilized entirely, the absorbance was measured using a microplate reader (Glomax discover, Promega) at 570 nm. To quantify the integrity of the cell membranes, the release of LDH into the growth media was measured after exposure to TPM. To this end, 50 µL of supernatant was incubated with a 50 µL well-mixed catalyst and dye substrate mixture (46:1) for 30 min at room temperature. The assay was terminated by adding 25 µL of stop solution, and the absorbance was measured using the microplate reader (Glomax discover, Promega) at 490 nm [23 (link)].

Overnight cultures of S. aureus/pSD1 and S. aureus/NADK sgRNA were diluted to an OD_600nm 0.05 in BHI supplemented with ATc (100 ng/mL) and chloramphenicol (7 μg/mL), spread in a 96 wells plate and incubated for 6 hr at 37°C with shaking at 200 rpm. After 5hr 30min of growth, lysozyme (5 mg/mL) and lysostaphin (0.05 mg/mL) were added. At t=6 hr, 100 μL of the CellTox Green Reagent (1:500, G8741, Promega) were added to bacteria. After incubation at RT for 15 min, both OD_600nm and fluorescent signal (excitation at 475 nm, emission at 500–550 nm) were measured with a microplate reader (Glomax Discover, Promega).
For the cytotoxicity assay on infected macrophages, culture medium was replaced by either DPBS or by 0.2% Triton X-100 6 hr after the infection. After 10 min at 37°C, cells were washed with DPBS, and then incubated with 100 μL of CellTox Green Reagent for 15 min at RT. Fluorescent signal (excitation at 475 nm, emission at 500–550 nm) was measured with a microplate reader (Glomax Discover, Promega).