| Title: |
PROTOCOL FOR: An optimized procedure for quantitative analysis of mitophagy with the mtKeima system using flow cytometry v1 |
| Authors: |
J Winsor, Nathaniel; A Killackey, Samuel; J Philpott, Dana; E Girardin, Stephen |
| Publisher Information: |
Springer Science and Business Media LLC |
| Publication Year: |
2020 |
| Description: |
Mitophagy is the process by which mitochondria are selectively targeted and removed via autophagic machinery to maintain mitochondrial homeostasis in the cell. Recently, the development of flow cytometry-based assays that utilize the fluorescent mtKeima reporter system have allowed for quantitative assessment of mitophagy at a single cell level. However, clear guidelines for appropriate flow cytometry workflow and downstream analysis are lacking, and studies using flow cytometry in mtKeima-expressing cells often display incorrect and arbitrary binary mitophagic or non-mitophagic cutoffs that prevent proper quantitative analyses. In this paper, we propose a novel method of mtKeima data analysis which preserves subtle differences present within flow cytometry data, in a manner that ensures reproducibility. Methods Summary:When used in conjunction flow cytometry, the fluorescent mtKeima reporter system facilitates the assessment of mitophagy at a single cell level. However, current experimental design and analysis methods inadequately capture biological shifts in measured cell populations. In this report, we propose an outline for the transfection of mtKeima into a cell monolayer, cell preparation and downstream analysis using flow cytometry and associated software, with emphasis on a novel data processing technique. |
| Document Type: |
other/unknown material |
| Language: |
unknown |
| DOI: |
10.17504/protocols.io.bimhkc36 |
| Availability: |
https://doi.org/10.17504/protocols.io.bimhkc36 |
| Rights: |
https://creativecommons.org/licenses/by/4.0/ |
| Accession Number: |
edsbas.84915219 |
| Database: |
BASE |