4.3. Imaging Autophagy with the RFP-GFP-LC3B Kit

MDA-MB-231 cells (1 × 10⁴ cells/well) were plated in a Nunc™ 177,437 Lab-Tek Chamber Slide System (Thermo Fisher Scientific, Rochester, NY, USA) and allowed to adhere overnight. The various stages of autophagy were monitored by the Premo™ Autophagy Tandem Sensor RFP-GFP LC3B Kit (Life Technologies, Carlsbad, CA, USA) according to the manufacturer’s instructions, which achieved efficient transduction using insect Baculovirus carrying the acid-sensitive LC3B-fluorescent protein chimera with a mammalian promoter. After the cells were treated overnight with 3 μL of BacMam reagents containing RFP-GFP-LC3B DNA, specified concentrations of drugs were subsequently added to the cell medium for 48 h. Aloperine (100 μM) was the positive control and induced autophagic flux, which was blocked by CQ. The media were removed, and live cell imaging solution containing Hoechst 33342 (1 µg/mL) was added and incubated for 20 min in the dark. The cells were then washed with 1× phosphate-buffered saline (PBS), covered in mounting medium (F4680) (Sigma-Aldrich, Inc. St. Louis, MO, USA), and imaged using a Zeiss laser scanning confocal microscope LSM800 (Carl Zeiss Microscopy GmbH, Jena, Germany).