Evos fl imaging system

HepG2 cells were transfected with GFP-LC3 plasmid. After 24 h, the cells were treated with CS3. Fluorescence of GFP-LC3-transfected cells was examined, and the images were generated via EVOS FL imaging system (Thermo Fisher, USA).
Immune-fluorescent assay was performed as described to further examine subcellular localization of LC3B in the HepG2 cells treated with or without CS3 13 (link),14 . In brief, HepG2 cells were also seeded on chamber slides (Thermo Fisher, USA) and treated with or without CS3 (20 and 40 μg/ml, respectively) for 24 h. The cells were washed three times with PBS, fixed with 4% paraformaldehyde for 15 min, and blocked with 10% goat serum in PBS. The cells were incubated with anti-LC3 antibody at 4°C overnight. After rinse three times with PBS, the slides were incubated with Alexa Fluor 555 secondary antibody at room temperature for 2 h in a dark container. The slides were rinsed with PBS and incubated with phalloidin (Alexa Fluor 488) for F-actin staining at room temperature for 2 h in a dark container. The slides were then rinsed with PBS and incubated with DAPI for nuclear staining in the dark for 30 min. After being washed with PBS and mounted with mounting medium. The slides were subjected to image examination via EVOS FL imaging system (Thermo Fisher, USA).

Human FOP iPSCs, BMSCs, or ASCs were plated at a density of 1 × 10⁴ cells/well in 24-well plate and 24 h later, they were incubated with rAAV1, rAAV2, rAAV2-TM, rAAV3, rAAV4, rAAV5, rAAV6, rAAV6.2, rAAV7, rAAV8, rAAV9, rAAVrh8, rAAVrh10, rAAVrh39, or rAAVrh43 vectors packaging the CBA-Egfp reporter transgene at three different titers (10⁹−10¹¹/mL genome copies). 48 h later, cells were washed with PBS and EGFP expression was monitored by the EVOS FL imaging system (ThermoFisher Scientific). Alternatively, cells were lysed in TNT lysis buffer and EGFP expression was assessed by immunoblotting with anti-EGFP antibody.

Bronchoalveolar lung organoids (BALO) were generated from flow-sorted murine lung epithelial stem cells co-cultured with flow-sorted mesenchymal cells as previously described^{36 (link)}. In lung organoid cultures, 3 × 10⁵ WT BMDM (1 or 2) or 4 × 10³ BMDM2 from Plet1^flx/flx or CX₃CR1^iCre-Plet1^flx/flx) and organoids were cultured in Matrigel® (Corning) (α MEM, 10% FCS, 100 U/ml penicillin, 0.1 mg/ml streptomycin, 2 mM L-glutamine, 1× insulin/transferrin/selenium, 0.0002% heparin)^{37 (link)}. In selected experiments, anti-Plet1 Ab (20 ng/ml) or isotype control (20 ng/ml) was added to the organoid medium upon cell seeding. BALO numbers and size were analyzed manually, by light microscopy with an EVOS FL imaging system (Thermo Fisher Scientific) and data are provided as means per each well with single data points representing one well.