Constanza Miguel if you could show the data image (ungated), it would have been better to understand data and give comment.

BTW you must be having technical data sheet with NAO, they must have explained the gating strategy as well.

When analyzing mitochondrial staining data by flow cytometry, the gating strategy can vary depending on the specific experiment and the markers used. Here are some general guidelines for gating in mitochondrial flow cytometry experiments:

Single-cell gating: Start by gating on single cells to exclude debris and cell aggregates. This can be done using forward scatter (FSC) and side scatter (SSC) parameters.

Mitochondrial mass: To analyze changes in mitochondrial mass, you can use a mitochondrial dye such as MitoTracker Green or MitoTracker Deep Red. Gate on the population with high fluorescence intensity for the mitochondrial dye to select cells with intact mitochondria

Mitochondrial membrane potential: To assess changes in mitochondrial membrane potential, you can use a dye such as tetramethylrhodamine, ethyl ester (TMRE) or JC-1. Gate on the population with high fluorescence intensity for the mitochondrial membrane potential dye to select cells with functional mitochondria

Superoxide production: To measure mitochondrial superoxide (ROS) production, you can use a dye such as MitoSOX Red. Gate on the population with high fluorescence intensity for the MitoSOX Red dye to select cells with increased ROS production

Mitochondrial DNA content: To study mitochondrial heterogeneity and mitochondrial DNA dynamics, a nanoscale, multi-parametric flow cytometry-based platform was used. In this case, mouse liver homogenates were prepared and MTG-labeled

Mitochondrial ROS production under hypoxia: In this case, a gate was drawn to exclude mitoSOX-negative cells and include mitoSOX-positive cells. This gating was then applied to any sample by drawing an icon of the "gate" on the corresponding plot

Salah A. Alshehade Thanks for your answer! I am interested in understanding how can you exclude debris from mitochondria using the parameters of FSC and SSC as both have the same size aprox. I used NAO to mark my mitochondrias.

Thank you very much!

You can try one of the following ways:

- Use a fluorescent mitochondrial dye like MitoTracker or NAO that stains only intact, active mitochondria. Debris and dead cells will not stain positively. Then you can gate on the fluorescence positive population.

- Use a viability dye like propidium iodide or DAPI to mark dead cells/debris. Gate out the viability dye positive population.

- Narrow the FSC and SSC gates to focus on the expected size and granularity of mitochondria. Debris tends to have lower FSC/SSC compared to intact mitochondria.

Try to include a purification step like centrifugation on a percoll gradient to separate mitochondria from debris after isolation. Stain the purified mitochondria population.

Also, optimize the mitochondrial isolation protocol to minimize debris - gentle homogenization, proper suspensions, low speed spins to remove nuclei/cell debris before collecting mitochondria.

@Salah A. Alshehade Thank you very much!! I ll do it