MitoSOX to MitoTracker Deep Red rates 3 channels

Parameters:

Name	#	Type	Description
Channel # for MitoTracker Deep Red	1	integer	Channel number associated with the image, as appears in the Multi-Dimensional Open Dialog. Use Edit/Rename to view or change channel number.
Channel # for TMRM	2	integer	Channel number associated with the image, as appears in the Multi-Dimensional Open Dialog. Use Edit/Rename to view or change channel number.
Channel # for MitoSOX	3	integer	Channel number associated with the image, as appears in the Multi-Dimensional Open Dialog. Use Edit/Rename to view or change channel number.
Background Level, Percentile	4	real	Background is calculated as frame-by-frame mean of those pixels that are the darker than this percentile of image histogram for the duration of the entire recording.
Mask sensitivity (0.2-1.5)	5	real	Sensitivity factor, typically 0.2-1.5
Frame range for rate and mean calculation	6	string	If left empty, all time points are shown, or give ranges to calculate means e.g. 1-3;7-9. Try also copy/paste marks in the plot window context menu.
Calculate rates for MitoSOX	7	boolean	Set this yes to calculate MitoSOX rate or no to calculate TMRM intensity

Description:

This protocol describes how to conduct mitochondrial a reactive oxygen species (ROS) measurement in adherent cells using fluorescence microscopy of MitoSOX (TPP-dihydroethidium). ROS levels are expressed as the rate of MitoSOX oxidation in %/sec normalized to MitoTracker Deep Red intensity (simple approach) or TMRM intensity (double normalization approach). The protocol is applicable to fluorescence microscopes capable of low-light level, time-lapse imaging. The normalization cancels the effects of differing cell size, density, geometry, and plasma and mitochondrial membrane potential-sensitive MitoSOX accumulation that are otherwise a major confounding factors of the MitoSOX assay. Microscope settings for recording the superoxide-specific OH-ethidium fluorescence is given below.
See detailed protocol at http://help.imageanalyst.net/protocols_MitoSOX.html

How to adjust the pipeline parameters:
*Channel #: Make sure that channel numbers listed here match the actual recording
*Background Level (percentile): Use a smaller background level (10-30 percentile) for confluent cultures and ~50 percentile for sparse cultures. Percentiles below 5-10 may increase noise.
*Mask sensitivity (0.2-1.5): This sets the amount of background masking. At higher values only the brighter parts of the cells are measured, and more background is omitted from the mean fluorescence intensity.
*Range for rate measurement: [important] Set the frame range where to measure rates and intensities, ideally from 1 to the last frame (e.g. 1-10).
*Calculate rates for MitoSOX: Normally yes, turn it off to see whether the increase of MitoSOX fluorescence is linear. (You can also toggle this in the context menu of the Plot Window).

After running the pipeline, in the Excel Data Window:
1) For each MitoSOX:MitoTracker Deep Red position calculate the ratio of Ch1_ROI1 and Ch3_ROI1 in an empty column
2) For each TMRM:MitoTracker Deep Red position calculate the ratio of Ch2_ROI1 and Ch3_ROI1 in an empty column
3) Finally, for each pair of sister cultures with MitoSOX and TMRM divide the two ratio values (form #1 and #2 above).
4) Save results in the Excel Data Window using the File/Save Excel Data Window main menu point.