mRFP-green fluorescent protein (GFP)-LC3 adenoviral vector

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	U937 cells were seeded in 24-well plates and differentiated into macrophages via induction by PMA. To detect autophagic flux, the macrophages were transfected with mRFP-green fluorescent protein (GFP)-LC3 adenoviral vectors according to the manufacturer’s instructions (Hanbio Inc., Shanghai, CN, USA). Successfully transfected macrophages could express LC3 protein tagged by RFP and GFP. GFP is acid sensitive and shows quenching green fluorescence in the acidic environment of a lysosome. However, in contrast with GFP, RFP is relatively stable within lysosomes. Therefore, the number of GFP and RFP puncta was examined and quantified by confocal microscopy. The 'red' and 'yellow' (i.e., a combination of red and green) spots indicated autophagosomes and autolysosomes, respectively.

Protocol tips

U937 cells were seeded in 24-well plates and differentiated into macrophages via induction by PMA. To detect autophagic flux, the macrophages were transfected with mRFP-green fluorescent protein (GFP)-LC3 adenoviral vectors according to the manufacturer’s instructions (Hanbio Inc., Shanghai, CN, USA). Successfully transfected macrophages could express LC3 protein tagged by RFP and GFP. GFP is acid sensitive and shows quenching green fluorescence in the acidic environment of a lysosome. However, in contrast with GFP, RFP is relatively stable within lysosomes. Therefore, the number of GFP and RFP puncta was examined and quantified by confocal microscopy. The 'red' and 'yellow' (i.e., a combination of red and green) spots indicated autophagosomes and autolysosomes, respectively.

Publication protocol

U937 cells were seeded in 24-well plates and differentiated into macrophages via induction by PMA. To detect autophagic flux, the macrophages were transfected with mRFP-green fluorescent protein (GFP)-LC3 adenoviral vectors according to the manufacturer’s instructions (Hanbio Inc., Shanghai, CN, USA). Successfully transfected macrophages could express LC3 protein tagged by RFP and GFP. GFP is acid sensitive and shows quenching green fluorescence in the acidic environment of a lysosome. However, in contrast with GFP, RFP is relatively stable within lysosomes. Therefore, the number of GFP and RFP puncta was examined and quantified by confocal microscopy. The 'red' and 'yellow' (i.e., a combination of red and green) spots indicated autophagosomes and autolysosomes, respectively.

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