Tandem mRFP-GFP-LC3 fluorescence microscopy

Cells were transfected with a plasmid containing mRFP-GFP-LC3 gene for 24h. Then the transfected cells were reseeded on glass coverslips in 6-well plates and cultured overnight. Subsequently, the cells were treated with culture medium with glutamine or not for 24h. The treated cells were fixed for 10 min by 4% formaldehyde and washed by PBS. The coverslips were transferred to glass slides and stained with diamidino-phenyl-indole (DAPI). The GFP/RFP signals were acquired by a Zeiss LSM 710 confocal microscope system (Carl Zeiss, Germany). Three different cells in SW480 and SW620 was counted the LC3 puncta.