Ethidium homodimer 1

The following procedure was adapted from our previous work^{12 (link)}. Cell viability was evaluated using 2 μM calcein AM (Invitrogen, green- live cells) and 4 μM ethidium homodimer-1 (Invitrogen, red - dead cells). After washing once with DPBS, the organoids were then imaged using the Olympus Fluoview Fv10i (Olympus, Tokyo, Japan) laser scanning confocal microscope.
Cell viability was evaluated using a Molecular Probe Live-Dead cell imaging system (Invitrogen).Human brain organoids were harvested for cell viability analysis at days 4, 5, 7, 10 and 21. Organoids were incubated at room temperature for 10 minutes in DPBS containing 2 μM calcein AM (Invitrogen, green- live cells) and 4 μM ethidium homodimer-1 (Invitrogen, red - dead cells). After washing once with DPBS, the organoids were then imaged using the Olympus Fluoview Fv10i (Olympus, Tokyo, Japan) laser scanning confocal microscope. The images obtained were then quantified using ImageJ to determine cell viability percentages.

Dopamine hydrochloride was purchased from Alfa Aesar (Gdansk, Poland). Doxorubicin hydrochloride (DOX·HCl) was purchased from LC Laboratories (Boston, MA, USA). PAMAM dendrimers G 3.0, phosphate buffer saline (PBS), Menadione, sodium tetraphenylborate, tris (hydroxymethyl) aminomethane, O-[N-(3-Maleimidopropionyl)aminoethyl]-O′-[3-(N-succinimidyloxy)-3-oxopropyl]heptacosaethylene glycol (molecular weight of 1570.76 g/mol), fetal bovine serum (FBS), penicillin, streptomycin, fibronectin, bovine collagen type I, bovine serum albumin, 0.1% poly-l-lysine, Muse^® Oxidative Stress Assay and MUSE^® Annexin V and Dead Cell assay were purchased from (Merck Darmstadt, Germany). Bronchial Epithelial Basal Medium (BEBM) and BEGM Bullet Kit were purchased from Lonza (Basel, Switzerland). WST-1 Cell Proliferation Reagent was purchased from Takara Bio (Shiga, Japan). Calcein AM, ethidium homodimer-1, Hoechst 33342, Minimum Essential Medium Eagle (MEM), dulbecco’s phosphate buffered saline (DPBS), 4% formaldehyde solution, sodium pyruvate, sodium pyruvate were purchased from Thermo Fisher Scientific (Waltham, MA, USA).

17-AAG (Selleckchem), Capsaicin (Cayman Chemical) and Ethidium Homodimer-1 (Thermo Fisher).

The live/dead staining kit contains two components. A dye calcein AM (Thermo Fisher) that permeates the plasma membrane and when cleaved by intracellular esterases fluoresces green and can be used to identify live cells. The second dye, ethidium homodimer–1 (Thermo Fisher), exhibits red fluorescence when intercalated into the DNA of dead or damaged cells. A549 cells (BCRC) were seeded in a 3.5 cm culture dish (Thermo Fisher) at a density of 8.5 × 10⁴ cells/dish and cultured for one day. The synthesized CaCO₃:Ce (1 mg/mL) in 2 mL of medium was added to each culture dish. After a 4 h reaction time, cells were irradiated with X-ray generated at 80 kV and 10 mA at a dose rate of 0.08 Gy/min for 100 s. All dishes were incubated at 37 °C in a 5% CO₂ atmosphere for one day. In the following two days, l mL of live/dead reagent prepared with phosphate-buffered saline (PBS, Thermo Fisher) was added into each dish and kept in the dark for 30 min, and then observed with a confocal microscope.

Bacterial cells were grown in Luria Bertani Broth (Sigma, Spain) and incubated in the absence or presence of 0.25× MIC of colloidal silver for 24 h as previously described [26 (link)]. The pellet was harvested by ultracentrifugation at 4600× g for 15 min. Bacterial cells were washed with phosphate-buffered saline (PBS) 1×, and, after centrifugation in the same conditions described before, the pellet was resuspended in 100 µL of PBS 1× containing 10 μL of ethidium homodimer-1 (EthD-1) (ThermoFisher, Spain). After 10 min of incubation, 100 μL of mixture was placed into a 96-well plate to measure fluorescence at 0, 5, 10, 20, 30, 60, 90, 120, 240, and 300 min using a Typhoon FLA 9000 laser scanner (GE Healthcare Life Sciences, USA) and quantified by ImageQuant TL software (GE Healthcare Life Sciences, USA).