Aug 20, 2025

MitoTracker staining of fibroblasts

DOI
https://dx.doi.org/10.17504/protocols.io.j8nlkyoo6g5r/v1
  • Ashley Seifert1,
  • Ebenezer Aryee1
  • 1University of Kentucky
Ashley Seifert
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DOI: https://dx.doi.org/10.17504/protocols.io.j8nlkyoo6g5r/v1
Protocol Citation: Ashley Seifert, Ebenezer Aryee 2025. MitoTracker staining of fibroblasts. protocols.io https://dx.doi.org/10.17504/protocols.io.j8nlkyoo6g5r/v1
License: This is an open access  protocol  distributed under the terms of the  Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: August 15, 2025
Last Modified: August 20, 2025
Protocol  Integer ID: 224797
Keywords: ASAPCRN, fibroblast, mitochondria, mitotracker, mitochondria in fibroblast, visualizing mitochondria, fibroblasts this protocol, fibroblast, seifert lab
Funders Acknowledgements:
Aligning Science Across Parkinson's
Grant ID: ASAP-020495
NIH
Grant ID: R01AR070313
Abstract
This protocol describes the procedure for visualizing mitochondria in fibroblasts with MitoTracker used in the Seifert Lab.
Protocol materials
DMEM and Hams F-12 50/50Fisher ScientificCatalog #MT10090CV
FBS (HyClone)
Antibiotic/Antimycotic (100X)Thermo Fisher ScientificCatalog #15240062
Tissue culture dish (10 cm)Merck MilliporeSigma (Sigma-Aldrich)Catalog #Z707651
HBSSThermo Fisher Scientific
MitoTracker® Red CMXRosCell Signaling TechnologyCatalog #9082
1X PBS
4% Formaldehyde, Methanol-FreeCell Signaling TechnologyCatalog #47746
AcetoneFisher ScientificCatalog #A18P-4
DAPI (4,6-diamidino-2-phenylindole, dihydrochloride)Thermo Fisher ScientificCatalog #D1306
HBSSGibco - Thermo Fisher ScientificCatalog #14170-112
Sterile deionized H2O
Prolong Gold Antifade MountantThermo Fisher ScientificCatalog #P36930
MitoTracker Green FMCell Signaling TechnologyCatalog #9047S
Seed fibroblasts in 24 well plates on cover slips at 5,000 cells per well
Culture cells in DMEM and Hams F-12 50/50Fisher ScientificCatalog #MT10090CV supplemented with 10% FBS (HyClone) and 1% Antibiotic/Antimycotic (100X)Thermo Fisher ScientificCatalog #15240062 at 37 °C under 3% O2 and 5% CO2 in Tissue culture dish (10 cm)Merck MilliporeSigma (Sigma-Aldrich)Catalog #Z707651 for 48:00:00

Remove media
Wash cells with warm HBSSThermo Fisher Scientific
MitoTracker Red
Follow this procedure for MitoTracker Red staining and fixed cell imaging
Add 0.3 μL/mL MitoTracker® Red CMXRosCell Signaling TechnologyCatalog #9082 in FBS-free DMEM for 00:30:00

Wash cells twice with 1X PBS

Fix for 00:15:00 in the dark with warm4% Formaldehyde, Methanol-FreeCell Signaling TechnologyCatalog #47746

Remove formaldehyde and wash cells twice with 1X PBS for 00:05:00

Add 500 µL chilled AcetoneFisher ScientificCatalog #A18P-4 to each well and incubate for 00:05:00

Remove acetone and wash cells once with 1X PBS
Add DAPI (4,6-diamidino-2-phenylindole, dihydrochloride)Thermo Fisher ScientificCatalog #D1306 diluted 1:2,000 in HBSSGibco - Thermo Fisher ScientificCatalog #14170-112 to each well and incubate for 00:15:00

Remove DAPI solution and wash cells with Sterile deionized H2O

Air dry coverslips
Mount on glass slides with Prolong Gold Antifade MountantThermo Fisher ScientificCatalog #P36930

MitoTracker Green
Follow this procedure for MitoTracker Green staining and live cell imaging
Prepare mix by adding 2 µL of MitoTracker Green FMCell Signaling TechnologyCatalog #9047S to 12 mL and warming

Incubate cells in staining mix in FBS-free DMEM for 00:30:00

Wash cells once with HBSSGibco - Thermo Fisher ScientificCatalog #14170-112

Live Image immediately. Cells should be cultured in Cellvis 24 Well glass bottom plate with high performance #1.5 cover glass # NC0397150 for optimal imaging.