Lysotracker dnd 26

The influence on mitochondria, lysosomes, and the ER was examined with immunofluorescence staining. Cells were seeded in Chamber Slides and stained after 72 h treatment. Then, cells were washed and the staining with MitoTracker Red CMXRos (20 nM, CellSignaling Technology, Danvers, MA, USA) and ER-Tracker Blue-White DPX (1 µM, Invitrogen) was done simultaneously for 35 min at 37 °C. Cells were washed and stained with LysoTracker DND-26 (50 nM, CellSignaling Technology) for 2 min at room temperature. Analysis was performed on a ZEISS Elyra 7 Confocal Laser Microscope (Zeiss).
Additionally, vacuole formation was analyzed after 72 h treatment using specific antibodies. Cells were harvested and incubated with Alexa488 anti-CD107a antibody (Biolegend, 1:50 in 0.1% BSA) for 30 min at 4 °C. Then, cells were washed and resuspended in 0.5 mL FluorFix^TM Buffer (Biolegend) for 20 min at room temperature. Afterwards, cells were washed twice with 1× intracellular staining perm wash buffer and incubated with Alexa594 anti-Rab7a antibody (Biolegend, 1:50 in 0.1% BSA) for 30 min at room temperature. The reaction was stopped with PBS and washed before cells were resuspended in 200 μL PBS (+2 mM EDTA). Cells were analyzed by flow cytometry on a Flow Cytometer (FACSAriaII, BD, blue (488nm) and yellow-green (561 nm) laser). Data analysis was performed using BD FACSDiva software (BD).

We used a previously described protocol (68 (link)) with modifications. Murine AT2 cells were isolated by dispersion of lungs with dispase and negative selection with antibodies against CD32/CD16 and CD45, followed by lamellar body staining with Lysotracker DND-26 (Cell Signaling Technologies [CST]) and pro-SPC polyclonal antibody (MilliporeSigma) to stain SPC.