Autophagy

Autophagy is a process in which eukaryotic cells use lysosomes to degrade their own cytoplasmic proteins and damaged organelles under the regulation of autophagy related gene (Atg). Under physiological conditions, autophagy levels are usually low. However, autophagy activity is significantly up-regulated under the stimuli of starvation, hypoxia and disease^[1].

Autophagy has different morphological characteristics in different stages, including early autophagy, middle autophagy and late autophagy. Microtubule-associated proteins light chain 3 (MAP1LC3), referred to as LC3 (light chain 3), run through the entire process of autophagy and are currently recognized as autophagy markers. In the process of autophagy, cytoplasmic LC3 (LC3-I) is enzymolysis of a small part of polypeptide, and is transformed into membrane type (LC3-II). Western Blot and fluorescence microscopy can detect the change of LC3-II/I ratio, and then analyze the level of autophagy^[1].

MCE has not independently verified the accuracy of these methods. They are for reference only.

The LC3 fluorescent staining of Hela cells was taken as an example

1. After starving the cells, remove the medium and add PBS to quickly wash the cells once.

2. Gently and rapidly add 2 mL of pre-cooled methanol into the cells and fix at -20℃ for 10 minu.

3. Remove methanol and gently add PBS for washing 3 times (cells can be temporarily stored at 4℃ PBS).

4. Block cells with buffer for 1.5 h.

5. Add LC3 antibody and incubate at 4℃ overnight.

6. Wash with PBS for 5 min (three times).

7. Add the corresponding fluorescent secondary antibody and incubate for 1 h at room temperature.

8. Wash PBS for 5 min (three times).

9. Add Hoechst and incubate for 10 min.

10. PBS washing for 5 min (three times) (can be temporarily stored in PBS or take fluorescence as soon as possible).

1. Fixation of samples

Cells should be fixed promptly after treatment to avoid protein degradation caused by delayed fixation.

2. Observation of results

Observation should be carried out in time after staining to avoid quenching of fluorescence;

If you are staining cell slides, you can use a mounting medium containing an antifade agent in the final step.