Figure 1
From: An improved method for high-throughput quantification of autophagy in mammalian cells
Construction of autophagy reporter cell lines. (A) Schematic overview. After random genomic integration of an eGFP-LC3B expressing cassettes into target cells by lentiviral delivery, single clones are isolated via FACS sorting and grown into clonal cell lines. (B) FACS sorting gating strategy for eGFP-LC3B-lentivirus transduced HeLa cells. Viable cell clones with intermediate eGFP-LC3B expression were chosen (blue). (C) Exemplary images of HeLa single cell clones expressing eGFP-LC3B in 96-well plates. Left panels: brightfield image and corresponding eGFP fluorescence (green) of a single clone after 1 week. Right panel, brightfield image and corresponding eGFP fluorescence (green) after 3 weeks, before transfer into larger wells. Size marker, 100 µm. (D) Detection of eGFP-LC3B expression in isolated clonal HEK293T, HeLa, Jurkat and THP-1 cell lines, comparing them to their respective parental cell line. Immunoblotting of whole cell lysates using anti-GFP, anti-LC3B and anti-β-actin antibodies. Uncropped western blots in Supplementary Fig. 3.
