Imaging Mitochondria

July 05, 2017

BlueskyReddit
Univesity of Texas Health Science Center
Translational & Vascular Biology
Assistant Professor

Overall

Quality of Results

Ease-of-Optimization

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Company:

Invitrogen (Thermo Fisher Scientific)

Product Name:

MitoTracker Deep Red

Catalog Number:

M22426

Among all fixation-stable mitotrackers, the deep red seems to be very selective and yields high contrast images. However, optimization for a incubation time and concentration is highly essential before using it in any experiments. Downside is not useful to visualize metabolically inactive mitochondrial structures.

Experimental Design and Results Summary

Application

Imaging mitochondria in cells

Starting Material

Cultured or adhered cells (e.g. human coronary artery endothelial cells), HBSS, PBS and MitoTracker Deep Red FM

Protocol Overview

Culture cells on a glass cover slip, after appropriate treatments or exposures, wash cells with warm HBSS or serum free medium. Then incubate with MitoTracker Deep Red prepared in HBSS or serum free incomplete medium for 2-15 min (first try concentration from 5 nM to 100 nM). General rule is metabolically highly active cells (such as muscle cells) require less time and low respiring cells (like endothelial cells) need more time with the dye. Then wash with warm HBSS twice and proceed with fixation using 4% parafarmaldehyde in PBS. FIxation is performed at 37C for 15 min. Now cells are ready for mounting and imaging. We have done IF staining after Deep Red treatment and obtained clear images. We did not test this reagent with suspension cells.

Tips

Warm serum free medium is better than complete medium. Its better to use higher concentration for shorter time to avoid circularization of mitochondria.

Results Summary

We treated human coronary artery endothelial cells with 100 nM mitoTracker Deep Red for 5 minutes in EBM-2 medium without serum. Washed three times with HBSS (dye free) and fixed as described above. Note the clear imaging of networked mitochondria in HCAECs. Imaged with 63x objective using Zeiss AxioObserver Z2 microscope. The image is pseudocolored with red.

DOI or PMID #

N/A

Additional Notes

N/A

Image Gallery

Summary

The Good

Quick method and yields high contrast images.

The Bad

Many conditions needs to be tried for optimization. Possible shape changes in mitochondria. Notable changes are circularizatiton and condensation.

The Bottom Line

This dye never failed with our cells. We have to optimize the concentration and incubation time for every cells. It does not stain in active mitochondria. Can not be used with FFPE sections.

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