I am trying to figure out how to use MitoSox green to measure the superoxide levels in human cerebral microvascular endothelial cells (HCMEC). For most ROS kits you can use a microplate reader to measure the amount of ROS expression with the cells. I wanted to use the MitoSox Green superoxide indicator as our microplate can only read up to 510 for excitation. The problem I am trying to figure out is that does the MitoSox Green work when using a plate reader. The protocol provided recommends using a 1 uM concentration, but the literature shows that many people using endothelial cells use a 5um concentration (for either Mitosox Green or Red). If I want to conduct the experiment at 5uM I have to use few wells as I only have 1 vial with 9mg of MitoSox that I have to dissolve in 10 uL to make a 1mM of stock before diluting further to solution. Do I use the recommended 1 uM or should I use concentration use by prior studies (5uM). The other aspect is imaging. Since I want to run this on a 96 well plate, I can image the wells using a flourescent microscope under a GFP filter. Can I use one plate to do both or should I do I plate for the plate reader and a chamber slide for the flourescent microscope. I do have a plan to do everything. I just want to make sure I am doing this correct unless someone has any advise to conduct this experiment better and more efficiently.
Mitosox, a fluorescent probe and derivative of dihydroethidium being conjugated with TPP+, enters mitochondria as the ptobe is +ively charged, and oxidized by superoxide and not by any other ROS or RNS. Oxidized Mitosox forms strong red fluorescence by binding with nucleic acid. As per literature, 5 micromole is the ideal concentration of Mitosox, but for flow cytometry 1 micromole is the preferred concentration as it is less prone to develop any interference in the reaction.
But Im using MitoSox Green not Red. Should it be the same concentration? Do I have to adjust it if I want to use a plate reader to measure the superoxide expression in the cells as well as use it for imaging?
We worked with MitoSOX Red at 5 µM for staining primary cultures of neurons and astrocytes. But we were using 6- and 24-well plates, which had a high number of cells and a large volume of medium.I believe you could definitely try using a lower concentration. The best approach would be to stain and image just one well as a test — that would quickly answer all your questions.Regarding your second question, yes, you can absolutely take images from wells within the same plate. In fact, this could give you an extra opportunity for internal comparisons later on.
Thank you Alina Chaplygina I plan to use a 4uM concentration to stain the cells. Because it's a 96 well plate Im thinking 24 hr incubation, 24 treatment, and then stain them. I'll image the wells first to have that as reference and then use the plate reader.