Hey Iseline,

we also use 5 µM MitoSOX and it worked for macrophages in a plate reader and in the FACS very well. But you are absolutely right, it seems your staining did not work. We stain our cells for both experimental setting as follows:

Wash cells once to remove medium and dead cells. Stain cells in HBSS with 5 µM MitoSOX f.c. for 15 mins at 37°C (this is way enough time for the MitoSOX to accumulate in the matrix). Remove the MitoSOX solution and wash two times with HBSS. Then continue with your prpearations and addtional stainings as you usually do. We use rotenone as positive control. It is a commonly used control for the MitoSOX probe. Rotenone blocks the Ubichinon-Bindingsite of complex I of the respiratory chain. So electrons get stuck in complex I, flow bacl into the matrix and ROS production in the matrix increases. So after rotenone treatment , if your cells are stained, you must get an increase of the signal. Perhaps this helps a little. You can have a look in our paper (with macrophages though) how it looks in a plate reader. So if you do not necessarily the FACS method a kinetic in the plate reader is always recommended, because ROS-SNapshots via FACS or microscopy do not show the whole picture.

https://stke.sciencemag.org/content/12/568/eaar5926 Figure 2D

All the best and keep me informed :)

Marc

Iseline Cardon

It is normal to have this kind of pattern. As there may be two kind of condition.

First, when you are getting single peak; overall all the cells are showing similar fluorescence intensity in a set, and that may vary to another set. Thus, you will notice single peak shift in one set to other.

Second, when you notice two peak; there is another condition where some portion of the cells in a set (suppose 50%) will show low fluorescence intensity and rest of the cell's fluorescence will increase. Thus, you will get two peaks.

Thank you for your replies.

It seemed like my staining did not actually work and only a few cells were correctly stained, resulting in these two peaks. When repeating with longer incubation of MitoSOX (30mn instead of 10mn), the peaks had a normal appearance and there was a clear shift between unstained and stained.

In context of this topic, I would like to bring to your attention a recent review of our group, "Functions of ROS in macrophages and antimicrobial immunity".

In this review, we give an introduction to ROS and their sources in macrophages, summarize the versatile roles of ROS in direct and indirect antimicrobial immune defense and provide an overview of commonly used ROS probes, ROS source inhibitors and ROS scavengers (also the difference between ROS scavengers and antioxidants, which are not synonymous, is explained).

If you like, please have a look at:

Functions of ROS in Macrophages and Antimicrobial Immunity

February 2021, Antioxidants 10(2):313

DOI: https://doi.org/10.3390/antiox10020313

Thanks Marc, I will definetely have a look