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Cell Cycle Analysis of ER Stress and Autophagy
A Popat1, A A Patel1, Gary Warnes2
1Flow Cytometry Core Facility, The Blizard Institute, Barts and The London School of Medicine and Dentistry, Queen Mary London University, London, UK.
Methods in Molecular Biology (Clifton, N.J.)
|September 10, 2022
Summary
Autophagy and ER stress, key cellular processes, can be measured using flow cytometry. This method quanties LC3B, PERK, protein misfolding, and reticulophagy, offering a cell cycle-dependent perspective.
Area of Science:
- Cellular biology
- Molecular biology
- Biochemistry
Background:
- Autophagy and endoplasmic reticulum (ER) stress are critical cellular processes.
- Western blotting is a common method for studying these processes, measuring markers like LC3B for autophagy and ER stress sensors (PERK, IRE1, ATF6).
- Sustained ER stress can lead to reticulophagy, a specific form of microautophagy targeting the ER.
Purpose of the Study:
- To explore alternative methods for measuring autophagy and ER stress.
- To investigate the cell cycle-dependent nature of autophagy and ER stress markers.
- To present flow cytometry as a viable technique for quantifying these cellular events.
Main Methods:
- Western blotting was used as a conventional approach.
- Flow cytometry was employed to measure LC3B upregulation, PERK activation, protein misfolding, and ER mass changes (reticulophagy).
- Antibodies and fluorescent probes for protein misfolding and ER tracking were utilized in conjunction with flow cytometry.
Main Results:
- LC3B, PERK, protein misfolding, and reticulophagy can be quantified using flow cytometry.
- These measurements can be performed in a cell cycle-dependent manner.
- Flow cytometry offers an alternative to Western blotting for assessing autophagy and ER stress.
Conclusions:
- Flow cytometry provides a powerful, cell cycle-aware method for evaluating autophagy and ER stress.
- This technique allows for the simultaneous measurement of multiple key markers.
- The findings suggest flow cytometry can complement or replace Western blotting in certain research contexts.
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