The observation and detection of cellular autophagy processes can be applied to (1) the study of cellular defense and stress regulation mechanisms; (2) the study of the problem of the origin of autophagosomal membranes. (3) The study of organelle autophagy.

Operation method

Tool drugs, fusion proteins, and other tracers of autophagy formation

Principle

The autophagic activity of normal cultured cells is very low and unsuitable for observation, therefore, autophagy must be artificially intervened and regulated, including autophagy inducers, autophagy inhibitors and other instrumental drugs, as well as antisense RNA interference technology (Knockdown), mutant strain screening, and exogenous gene introduction. and detection by techniques such as fluorescence microscopy or Western Blot.

Materials and Instruments

Cell
Autophagy inducer Autophagy inhibitor Monodansulfonylcadaverine (MDC) 0.1% SDS
Fluorescence microscope Cell crawler

Move

i. The autophagy activity of normal cultured cells is very low and not suitable for observation, therefore, autophagy must be artificially interfered and regulated.

II. After cells are induced or inhibited, the autophagy process needs to be observed and detected, and the commonly used strategies and techniques are:

1. Observe the formation of autophagosomes;

2、Tracing autophagy formation by using GFP-LC3 fusion protein under fluorescence microscope;

3, Detecting changes in the LC3-II/I ratio using Western Blot to evaluate autophagy formation;

4, Detection of bulk degradation of long-lived proteins;

5, MDC (Monodansylcadaverine, monodansylcadaverine) staining;

6, CellTrackerTM Green staining: mainly used for double staining, but it can stain all vesicles, so it is also non-specific;

Caveat

I. Autophagy inducers1. Bredeldin A / Thapsigargin / Tunicamycin: mimics endoplasmic reticulum stress2. Carbamazepine / L-690, 330 / Lithium Chloride: IMPase inhibitor (i.e. Inositol monophosphatase)3. Earle's Balanced Salt Solution: creates starvation4. N-Acetyl-D-sphingosine (C2-ceramide): Class I PI3K Pathway Inhibitor5. Rapamycin: mTOR inhibitor6. Xestospongin B/C: IP3R blockerII. Autophagy inhibitors1.3-Methyladenine (3-MA): (Class III PI3K) hVps34 inhibitor2. Bafilomycin A1: proton pump inhibitor3. Hydroxychloroquine: Lysosomal lumen alkalizerIn addition to the selection of the above tools, it is usually necessary to intervene in autophagy-related genes in combination with genetic techniques: including antisense RNA interference technology (Knockdown), mutant strain screening, and exogenous gene introduction.

Common Problems

When studying autophagy-related proteins, their localization is required. Since autophagosomes are closely related to lysosomes, mitochondria, endoplasmic reticulum, and Golgi apparatus, some tracer proteins are commonly used to co-localize them under fluorescence microscope in order to differentiate them:

Lamp-2: Lysosomal membrane protein, which can be used to monitor the fusion of autophagosomes with lysosomes.

LysoTrackerTM probe: available in red or blue, shows all acidic vesicles.

pDsRed2-mito: vector, transfected to express a fusion protein (red fluorescent protein + mitochondrial matrix localization signal) that can be used to detect the extent to which mitochondria are being autophagocytosed (Mitophagy).

MitoTraker probe: specifically displays live mitochondria and fluorescence is retained after immobilization.

Hsp60: localizes to the mitochondrial matrix and is not released upon cell death.

Calreticulin (calcium reticulin): endoplasmic reticulum lumen

(Note: These proteins are all cytoplasmic proteins, crawl or trypsin-digested cells need to be permeabilized before doing immunofluorescence, which can be treated with 0.1% SDS.)

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