Hi,
I am planning on performing MitoSox, MitoTracker Green and Mitochondrial Membrane potential assay using a plate reader (SpectraMax M3). My Mitosox protocol is the following: 24h incubation, 24h drug treatment, wash with DPBS, incubate 10 min at 37C with 5uM MitoSox diluted in DPBS, wash one time, add fresh buffer and read at 518/580nm. I am using 5uM Doxorubicin as a positive control. My question is the following: the results of my first attempt looked weird with really different values within the same replicates and higher values in the Non treated control compared to positive control - Does anyone know how to set the plate reader? Do we need to use "endpoint"/"kinetic" or "spectra scan" reading? Thanks !
Hi Soraya
I am not very familiar with MitoSox but I would like to make a few general remarks.
If you use a plate reader you will always take the reading from a specific location in the plate you shold be able to figure out how large the region is and where it is localised in the well. Considering this you will find that you will have to have a very consistent culture e.g. always confluent in the reading area.
You also will have to make sure that the reader is focussing on the bottom of the wells where the cells are located. Different plates have different skirt height so the focus position of the reader should not be off. You will probably find instructions for this in the manual?
If possible confirm your cell seeding on a microscope and - if you have access to a fluorescene microscope also confirm the staining best with some images.
If you stain in DPBS at 37°C the cells will basically not have any nutrients during this time? This will change a number of things in their energy metabolism for this period and will influence the uptake of the dye.
best
Heiko
- Badges
- Science topic
- Similar topics
- Phytochemistry
- Extracts