Pi rnase staining buffer

HeLa, HepG2, and HCT116 cells were costained with LysoTracker Red (0.3 µM) and Hoechst 33342 for 30 min. After extensive washing, cells were suspended in cold PBS and analyzed by flow cytometry. For LAMP1 immunostaining, cells were first fixed with 4% PFA for 15 min and permeabilized with permeabilization buffer (1× PBS, 0.3% Triton X-100, and 0.5% BSA) for 15 min and incubated with LAMP1 antibody in the incubation buffer (0.5% BSA in PBS) for 1.5 h at room temperature. After extensive washing, cells were resuspended in incubation buffer containing Alexa Fluor 488 secondary antibody for 30 min. Cells were then collected and incubated in PI/RNase staining buffer (4087; Cell Signaling Technology) for an additional 30 min and analyzed by flow cytometry. For the FITC-Dextran experiment, cells were fed with 0.25 mg/ml FITC-Dextran for 12 h followed by 2-h recovery in fresh DMEM medium. Cells were then fixed with 70% ethanol (2 h, 4°C) and stained with PI for 30 min and analyzed by flow cytometry. All samples were analyzed on a FACSAria SORP machine (BD Biosciences), and the fluorescence intensity profiles of each cell cycle stage were analyzed using BD FACSDiva 8.0.1. Cell cycle distribution was analyzed by PI or Hoechst 33342 content and fitted to the Dean-Jett-Fox cell cycle model using FlowJo software.