Jan 23, 2025
Flow cytometry measurements of lipid peroxidation with C11-Bodipy 581/591
- 1UCL / The Francis Crick Institute
Protocol Citation: Laura Mahoney 2025. Flow cytometry measurements of lipid peroxidation with C11-Bodipy 581/591. protocols.io https://dx.doi.org/10.17504/protocols.io.dm6gp92x1vzp/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: January 23, 2025
Last Modified: January 23, 2025
Protocol Integer ID: 118961
Keywords: flow cytometry, Lipid peroxidation
Abstract
Protocol for staining iPSC-derived neurons for lipid peroxidation measurements using Live/Dead and C11-Bodipy 581/591 dyes, via flow cytometry
Materials
| A | B | C | |
| BODIPY 581/591 C11 | Thermo Fisher Scientific | Cat#D3861 | |
| LIVE/DEAD Blue | Thermi Fisher Scientific | Cat#L23105 | |
| Accuase | StemCell Technologies | Cat#07922, 07920 | |
| PBS, pH 7,4 | ThermoFisher | Cat# 10010023 |
Cell treatment
Cell treatment
Treat iPSC-derived neurons platted in 24-well plates with 300-400 µL of RSL3 in normal culture media for 3 to 5 hours
Cell staining with Live Dead Blue and C11-Bodipy 581-591
Cell staining with Live Dead Blue and C11-Bodipy 581-591
1h 10m
1h 10m
Remove the media from the wells and transfer into 1.5ml eppendorf tubes, in order to collect dead cells.
10m
Add 300 µL of pre-warmed accutase to each well and return plate to the incubator for00:10:00 to 00:15:00 incubation at 37 °C
15m
Pippette accutase up and down (carefully not to make bubbles), in order to dissociate the cells into a single cell suspension, and transfer the cells into the appropriate 1.5ml eppendorf tubes.
5m
Centrifuge cells at 300 x g, 20°C, 00:05:00
5m
Aspirate the supernatant without disturbing the pellet.
Resuspend the cell pellet with 100 µL of Live/Dead Blue (Cat#L23105) diluted in PBS: 0.5 µL per 1 mL of PBS (Note: concentration to be determined for each cell model).
5m
Place cells in a 37 °C incubator for 00:15:00
15m
Remove cells from incubator and add 100 µL of C11-Bodipy 2x concentrated to the cells, so the final concentration is 2 micromolar (µM)
Place cells in a 37 °C incubator for 00:15:00
15m
Remove samples from the incubator and place in ice. Sample is ready to take to Flow cytometer
Data acquisition
Data acquisition
Adjust gates in order to acquire FITC and PE Texas Red in the live, single cell population.
Acquire data from at least 10,000 single live cells.
To report lipid peroxidation, the ratio of oxidised C11-Bodipy (em: 488nm) over non-oxidised C11-bodipy (em:568nm) was measured and normalised to control population.