Transduction with bacmam RFP-GFP-LC3B construct to detect autophagic flux

Formation and maturation of autophagosomes was monitored in control and SLOS RPE cells by transient transduction utilizing a Premo™ Autophagy Tandem Sensor RFP-GFP-LC3B Kit (ThermoFisher Scientific, {"type":"entrez-protein","attrs":{"text":"P36239","term_id":"548754"}}P36239) as described by the manufacturer. Briefly, RPE cells cultured on RINZL coverslips were incubated with 30 particles/cell of the BacMam reagent containing the RFP-GFP-LC3B construct (n = 3), for 24 h in the cell culture incubator as described above. Transduced control RPE cells (n = 3) were incubated in 150 µM CHQ for 8 h. The cells were rinsed in 1X PBS followed by 4% buffered paraformaldehyde fixation for 10 min. Fixed coverslips were then rinsed, and counterstained with DAPI. Coverslips were mounted using Vectashield® mounting medium, and high magnification confocal fluorescence microscopy images were obtained as described in Section 4.7. Transduced cells were selected for microscopy based on RFP signal alone and RFP/GFP signals were subsequently captured. The number of GFP- and RFP-positive puncta were manually counted (n = 100/group/treatment), and the GFP/RFP ratio provided an estimate of the percentage of unacidified (immature) phagosomes under each condition.