MitoSOX:MitoTracker Deep Red Ratio End Point with Post-hoc ICC-based Gating

This pipeline measures superoxide levels by calculating MitoSOX:MitoTracker Deep Red (MTDR) Ratio in a subpopulation of cells in a view field, marked by post-hoc immunocytochemistry (ICC). In addition the fraction of live cells (based on MitoTracker Deep Red uptake) and the fraction of ICC positive cells can be calculated from the recorded cell counts.

The input (two recordings):
Live recording: MitoSOX, MitoTracker Deep Red, Hoechst, three channel recording. Tag the Multi-Dimensional Open dialog as #1.

Staining and imaging protocol:

1) Incubate cell in culture medium with MitoSOX 2uM + MitoTracker Deep Red 10nM for 30-60min.
2) Replace culture medium with a clear medium containing no MitoSOX and MitoTracker and supplemented with MnTMPyP 20uM, Hoechst 33342 5ug/ml, BSA 0.4%
3) Capture three-channel live cell images within 30min (the MitoSOX fluorescence is relatively stable in the presence of MnTMPyP).

Post-hoc immunocytochemistry: coarsely the same view field imaged at two channels: immunofluorescence and Hoechst. Tag the Multi-Dimensional Open dialog as #2.
1) Staining and imaging protocol: use green fluorescent label, because MitoSOX and MitoTracker Deep Red fluorescence will be retained.

Notes on the pipeline:
The pipeline subtracts background, registers channels (for tiled images for each tile) and registers the shift between the live cell and post-hoc ICC images. The pipeline corrects for crossbleed between the MTDR and MitoSOX channels. This pipeline does not remove debris in MitoSOX channel based on morphological criteria (in contrast to the “MitoSOX:MitoTracker Deep Red Ratio End Point” pipeline), but instead manual masking can be used by a pre-defined ROI file. After this image preparation, the nuclear marker Hoechst image is used as seed to determine total cell count and to segment the MTDR image. Cells brighter than “Minimal MitoTracker intensity of cells (% of maximum)” are defined as live cell. Then those MTDR segments are selected as ICC positives (and also live) that are brighter than ”Intensity threshold for ICC staining (% of maximum)”. Finally the MitoSOX and MitoTracker Deep Red images are masked so measure the average intensities of the two probes only in the ICC positive, live cells.
Tiled images: the background tiling pattern is efficiently removed by spatial filtering if the recording was performed without overlap and image registration. Provide the number of tiles in x and y direction.

Excel output:
Ch1: Average MitoSOX intensity in the ICC positive, live cells
Ch2: Average MitoTracker Deep Red intensity in the ICC positive, live cells
Ch3: Count of nuclei (all cells, alive and dead)
Ch4: Count of live cells (MitoTracker Deep Red positive)
Ch5: Count of ICC positive, live cells

Notes on pipeline parameters:
“ROI File to manually mask debris” if a ROI file is given here, the area covered by the ROIs will be masked.
“Spectral Unmixing Coefficient Matrix:” This corrects for the bleedthrough of MTDR into the MitoSOX channel, which is usually very low. To determine spectral crossbleed coefficients use the Tools/Calculate Crossbleed Correction Factor main menu point, or the Math/Blind Spectral Unmix with NMF function. See more on the layout of the Spectral Unmix Coefficient Matrix in the description of Math/Spectral Unmix. Enter MitoSOX as probe/channel #1 and MTDR as probe/channel #2 into the spectral unmix coefficient matrix.
”Intensity threshold for positive staining (% of maximum)”: Cells are determined as positive for the immunofluorescence if are brighter than this percent of the maximal fluorescence of the post-hoc immunofluorescence image. The maximal fluorescence is not the intensity of the brightest pixel, determined at a percentile level, which is given by the “Debris cutoff (percentile)” parameter.

Version history
V2:
Layout of data columns as channels 1,2,3,4,5 has been fixed
Warning for the use of reference images have been added