Zen 3.2 blue edition

After removal of media, the cells were washed by PBS, and fixed with 4% formaldehyde for 15 min at room temperature. After one PBS wash, cells were permeabilized by 0.2%Triton-X100 in PBS for 20 min, washed by PBST solution (0.1% Tween-20 in PBS pH 7.4) three times, and blocked by 3% BSA (PBST solution) for 1 h at room temperature. With three PBST washes between each incubation step, the cells were incubated with primary antibody rabbit anti-AIF mAb (Abcam, ab32516, 1:500 dilution) combined with mouse anti-V5 mAb (Biodragon, B1005, 1:1000 dilution) at 4 °C overnight, followed by incubation fluorescent antibodies goat anti-Rabbit-AlexaFluor488 (Invitrogen, A-11008, 1:500 dilution) combined with goat anti-Mouse-AlexaFluor555 (Invitrogen, A-21422, 1:500 dilution) and Hoechst 33342 (2 μg/mL) at room temperature for 1.5 h. After three PBST washes, the samples were subjected to microscopy analysis on an LSM 700 laser scanning confocal microscope (Zeiss). The fluorescent images were captured in the Hoechst channel (λ_ex = 405 nm), AF488 channel (λ_ex = 488 nm), and AF555 channel (λ_ex = 555 nm). Processing of images were carried out using ZEN 3.2 blue edition (ZEISS). Pearson’s R values for colocalization were calculated for individual cells using the Coloc2 tool embedded in Fiji-Image J (v1.53k).

Approximately 10,000 HeLa cells were seeded in a LabTek-II 8-well glass chamber with 200 µL DMEM (+10% FBS) and incubated at 37 °C with 5% CO₂ for 24–48 h. The medium was removed and the cells were rinsed with PBS. Ir1 (0.5 μM) was combined with MitoTrackerTM Deep Red (0.1 μM, Thermo) in 100 µL DMEM and the cells were incubated with the mixture for 30 min at 37 °C. After washing by PBS three times, the cells were immediately analyzed o an LSM 700 laser scanning confocal microscope (Zeiss). The fluorescent images were captured in the Hoechst channel (λex = 405 nm) for photocatalyst luminescence and the Cy5 channel (λex = 639 nm) for MitoTracker using a 63× oil lens. Processing of images were carried out using ZEN 3.2 blue edition (ZEISS). Pearson’s R values for colocalization were calculated for individual cells using the Coloc2 tool embedded in Fiji-Image J (v1.53k).

Lungs were collected from indicated mice sacrificed on day 6 post-infection. Tissues were covered in cryo-embedding media, kept in liquid nitrogen until completely submerged, then stored at −80 °C until ready for sectioning. 4 mm sections were cut with a cryotome and stained with haematoxylin and eosin. For immunofluorescence, sections were fixed in 4% paraformaldehyde for 1 h at RT, washed with PBS, then incubated in “permeabilisation solution” containing 0.05% TX-100 and 0.1% sodium citrate for 2 min on ice (4 °C). Cell death was analyzed with an in situ cell death detection kit (TMR-red, Roche) after antigen retrieval either alone or followed by staining with anti-CD45 (AB 10558 Abcam) and Goat anti-Rabbit IgG DyLight488 (ThermoFicher ref: 35553). Anti-CD8a (AB 217344, Abcam) was used 1/100 combined with a secondary Goat anti-Rabbit IgG AF568. Hoechst 33342 was used to stain nuclei. Brightfield and fluorescence microscopy was performed using ZEISS Axio Scan.Z1. Samples were analyzed with Zen 3.2. blue edition (Zeiss) and quantified with QuPath software 0.2.3.