Feb 26, 2025
Lipid ROS measurements via high-throughput imaging
- 1Gandhi Lab
Protocol Citation: Alexis Penverne 2025. Lipid ROS measurements via high-throughput imaging. protocols.io https://dx.doi.org/10.17504/protocols.io.5qpvo9ek9v4o/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: February 26, 2025
Last Modified: February 26, 2025
Protocol Integer ID: 123440
Abstract
Lipid ROS measurements via high throughput imaging on neurons using C11-BODIPY dye
Cells cultured in PLO and laminin in 96-well microplate. From day 14 after neuronal induction, cells are ready to be imaged.
Stain cells with 2 micromolar (µM) C11-BODIPY 581/591 in HBSS for 00:15:00 at 37 °C
15m
Remove probe by washing with HBSS and treat with 20 micromolar (µM) RSL3+/-
Immediately after treatment, image cells on High Content Imaging system (Operetta), confocal setting with 20x objective. A minimum of 9 fields of view and 6 z-slices per well are acquired for a total of 06:00:00 (acquisition every00:30:00 for each well).
An image analysis pipeline was used to detect cells with reduced (red) or oxidised (green) signal. Lipid ROS levels are determined by calculating ratio of oxidised over reduced.