Quantifying Autophagy in Cell Lines

HeLa or HEK293T eGFP-LC3B reporter cells were grown on coverslips (VWR) in 24-well plates and treated as indicated. THP-1 or Jurkat eGFP-LC3B reporter cells were grown in 24-well plates and treated as indicated. Cells were fixed with 4% (w/v) paraformaldehyde (PFA, Santa Cruz) for 20 min at RT, permeabilized with 0.5% (v/v) Triton-X-100 (Sigma-Aldrich) in PBS and then blocked with 5% (v/v) fetal bovine serum (Gibco) in PBS for 1 h at RT. Adherent cells were mounted on microscope slides (VWR) in 4′,6-diamidino-2-phenylindole (DAPI, VWR)-containing Mowiol mounting medium (Mowiol 4–88 10% (w/v, Carl Roth), glycerol 25% (w/v, Sigma-Aldrich), H₂O 25% (v/v), 0.2 M Tris HCl pH 8.5 50% (v/v, AppliChem GmbH, VWR), DABCO 2.5% (w/v, Carl Roth)^{10 (link),38 (link)}) to co-stain nuclei. The suspension cells were pelleted (300 g, 3 min), suspended in the same mounting medium, and mounted between coverslips and microscope slides (VWR). All laser scanning images were acquired on a Zeiss LSM 710 confocal microscope. The total area of cytoplasmic eGFP-LC3B puncta in HeLa, HEK293T, Jurkat and THP-1 cells was determined using a custom ImageJ macro for ≥ 30 randomly selected cells.