U dish

Pseudomonas. aeruginosa cultures incubated for 24 h were adjusted to an OD600 of 0.8 and then diluted 1:100, and 1 ml of the prepared broth was inoculated into ibiTreat petri dish (ibidi, Munich, Germany) with 1 ml of the Nissle cell-free supernatant. The dish was incubated for 24 h at 37°C. 1 ml of P. aeruginosa inoculated with 1 ml of its supernatant was used as a negative control. For assessing effects on mature biofilms, an overnight broth of P. aeruginosa was adjusted to OD600 of 0.8 and then diluted 1:100 and 1 ml of the prepared broth was inoculated into u-Dish (ibidi) and incubated for 24 h at 37°C. After incubation, the planktonic cells were removed and 1 ml of Nissle cell-free supernatant was added and then incubated for another 24 h. 1 ml of P. aeruginosa supernatant was used as a negative control.

To evaluate the mitochondrial network and dynamics DIM-induced hPADs isolated from each experimental group, were cultured on 35 mm u-Dish (Ibidi GmbH, Munich, Germany) for 10 days and then stained with 200 nM MitoTracker Green^FM (Invitrogen Life Technologies, Carlsbad, CA), as previously described [8 (link)]. Cells were imaged, with time lapses recorded for 3 min with time intervals of 10 s, using an inverted Leica DMI6000 microscope (Leica, Wetzlar, Germany) equipped with a stage incubator (Pecon, Erbach, Germany) with controlled temperature, humidity and CO₂.
Superoxide radical production in hPADs was monitored by dihydroethidium (DHE; Invitrogen) fluorescence and quantified by measuring the change in fluorescence intensity in the nuclei of rPAD cells during imaging, as previously described [8 (link)].
Images were captured through a 63 × 1.2NA water immersion objective, with a DFC350FX camera and Leica filter set L5 (for MitoTracker Green), and N2.1 and N3 (for DHE). All image analyses were performed using Fiji ImageJ software [46 (link)].