Slide 8 well ibitreat chambers

Typically, 20,000 HeLa cells in 200 µL were seeded on µ-Slide 8 well-ibiTreat chambers (1 cm²per well, Ibidi, Germany) at least 12 h before the particle exposure. UCNPs were diluted to a concentration of 2.5 nM in cell media. After 3 h, cells were cleaned with PBS (3×) in order to remove non-associated particles. 200 µL of supplemented DMEM without phenol red were added to the cells before imaging on an Andor Dragonfly spinning disk confocal system mounted on a Nikon TiE microscope equipped with a Zyla 4.2 PLUS camera (Andor, Oxford Instruments) and an OKO-lab incubator to keep cells at 37 °C during experiment time. RB channel was obtained with an excitation of a 561 nm laser and a 620/60 nm filter. Ce6 emission was collected exciting with a 405 nm laser and using a 725/40 nm filter. All the images were processed with the free software ImageJ. For colocalization experiments lysosomes were labeled with lysotracker blue (Ex. 405, Em. 450/50, Thermofisher) and Mitotracker Green FM (Ex. 488, Em. 525/50, Thermofisher) following the manufacturer instructions.

Organoids were expanded in µ-Slide 8-well ibiTreat chambers (Ibidi, Fitchburg, WI) and treated with 200 nM camptothecin (CPT) for 24 h. As a control, some wells were left untreated. Phosphorylation of the Ser-139 residue of the histone variant H2AX (γH2AX) was assessed by immunofluorescence staining following already published protocols (Mayorgas et al., 2021 (link)), with some modifications. After the fixation step, organoids were stored in PBS overnight at 4°C, and the next day the permeabilization and blocking steps were performed as indicated. Samples were then incubated with anti-γH2AX (#ab81299, 1:750, Abcam) and EpCAM (#M0804, 1:150, Dako) primary antibodies overnight at 4°C. The next day, samples were incubated with the secondary antibodies anti-rabbit Alexa Fluor^® 594 and anti-mouse Alexa Fluor^® 488 (ThermoFisher, Waltham, MA), both diluted 1:500. After DAPI nuclear staining, samples were overlaid with Ibidi Mounting medium (#50001, Ibidi, Fitchburg, WI) and stored at 4°C for subsequent fluorescent microscope observation in a Zeiss LSM 880 confocal laser scanning microscope (CCiTUB optical microscopy facility, Universitat de Barcelona, Spain). Positive nuclei (γH2AX–DAPI colocalization) were counted by using the CellCounter plug-in in ImageJ software. The ratio of γH2AX-positive nuclei versus the total number of nuclei per organoid was calculated.