The genes for autophagosome- and mitochondria-specific proteins were linked to fluorescent protein genes to form fusion genes, which were introduced into cells for expression, and the tagged proteins were tracked in vivo intracellularly with the aid of fluorescence microscopy or confocal microscopy, which enabled co-localization of autophagosomes and mitochondria for visualization and analysis.

Appliance

Observing the dynamics of mitochondrial autophagy.

Operation method

Co-localization of autophagosomes and mitochondria by immunofluorescence

Principle

The genes for autophagosome- and mitochondria-specific proteins were linked to fluorescent protein genes to form fusion genes, which were introduced into cells for expression, and the tagged proteins were tracked in vivo intracellularly with the aid of fluorescence microscopy or confocal microscopy, which enabled co-localization of autophagosomes and mitochondria for visualization and analysis.

Materials and Instruments

Cells, 12-well plate
Autophagosome-specific protein EGFP-LC3
Mitochondria-specific protein DsRed-mito
Serum-free medium, PEI transfection reagent
Fluorescence microscope or confocal microscope

Move

1. Plate spreading: Trypsin digestion of cells in logarithmic growth phase was performed in 12-well plates, and the amount of plate spreading should be 70~80% when transfection was performed on the next day.

2. Transfection: Prepare two 1.5 mL EP tubes, add 50 μL of serum-free medium, add appropriate amount of EGFP-LC3 and DsRed-mito plasmid to one tube, and add 3 times the volume of PEI transfection reagent to the other tube (amount of DNA plasmid: PEI = 1:3), and then mix the liquids of the two EP tubes after 5 min of standing at room temperature, and then stand at room temperature for 15 min. After that, add the transfected complex into the cells slowly drop by drop, and replace the medium with complete medium after 4~6 hours.

3、Observation of fluorescence: 24 h after transfection, the fluorescence can be observed under fluorescence microscope or confocal microscope. In the same field of view, observe the green fluorescence of autophagosome EGFP-LC3 under blue excitation light and the red fluorescence of mitochondria DsRed-mito under green excitation light, and then use DAPI to stain the nucleus and observe the blue fluorescence of the nucleus under UV-excitation light if necessary. Finally, Images were merged to observe the co-localization of autophagosomes and mitochondria.

Caveat

1, serum has a great influence on the transfection efficiency, the cells should be transfected with complete medium (without antibiotics), and serum-free medium should be used to dilute plasmid DNA and transfection reagents.

2， the cell state is very critical, the cell state is best when the cells are resuscitated for about 3 generations, try not to use the cells that have been passed for many generations.

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