Direct co-staining is not recommended.

The core reason is consistent with the previous analysis:

Serious spectral overlap:

The emission light of TMRE (excitation/emission about 554/578 nm) and MitoSOX Red (excitation/emission about 510/580 nm) are both red, which is difficult to completely separate through filters in fluorescence microscopy or flow cytometry, and easily leads to signal cross-interference.

Interference of biological effects:

TMRE depends on mitochondrial membrane potential (ΔΨm) to enter mitochondria, and although the uptake of MitoSOX Red does not directly depend on membrane potential, its fluorescence intensity after oxidation may be affected by the functional state of mitochondria (such as membrane potential changes, oxidative stress levels), and simultaneous staining of the two may interfere with each other's detection accuracy.

Technical points if co-staining is necessary (strict optimization conditions are required)

1. Staining order and time optimization

Stain MitoSOX Red first: Because its superoxide detection process is faster (usually staining for 10-15 minutes), and the oxidation reaction is an instantaneous event, you can complete the staining and washing first, and then perform TMRE staining.

Interval staining and washing: After MitoSOX staining, wash thoroughly with serum-free medium or PBS (2-3 times) to avoid residual dye affecting TMRE binding; TMRE staining time is usually 15-30 minutes, and the cell state must be stable during staining.

2. Dye concentration and incubation conditions

Reduce dye concentration: For example, the common concentration of TMRE is 10-20 nM, and MitoSOX Red is 5-10 μM. You can try to decrease the concentration by 1/2 or 1/3 to reduce the intensity of fluorescence overlap.

Temperature and light-proof control: Keep a constant temperature of 37℃ during the staining process to avoid low temperature affecting mitochondrial function; incubate in the dark to prevent dye photobleaching or nonspecific oxidation.

3. Adjustment of fluorescence detection channels

Multi-channel time-segment acquisition: If a laser confocal microscope is used, different excitation wavelengths can be set (such as 561 nm laser for TMRE and 488 nm or 514 nm laser for MitoSOX), and signals can be collected under long-pass or band-pass filters (such as 580-650 nm for TMRE and 590-650 nm for MitoSOX) to minimize spectral overlap.

Flow cytometry parameter analysis: Use the dual fluorescence channels of the flow cytometer (such as PE and APC channels) to detect the signals of both, but the compensation parameters need to be determined through the single-staining control group to avoid fluorescence leakage.

References-PMID: 37246868.

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