Aug 21, 2025

Public workspaceMitoTracker 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 16, 2025
Last Modified: August 21, 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
ReagentDMEM and Hams F-12 50/50Fisher ScientificCatalog #MT10090CV
ReagentFBS (HyClone)
ReagentAntibiotic/Antimycotic (100X)Thermo Fisher ScientificCatalog #15240062
ReagentTissue culture dish (10 cm)Merck MilliporeSigma (Sigma-Aldrich)Catalog #Z707651
ReagentHBSSThermo Fisher Scientific
ReagentMitoTracker® Red CMXRosCell Signaling TechnologyCatalog #9082
Reagent1X PBS
Reagent4% Formaldehyde, Methanol-FreeCell Signaling TechnologyCatalog #47746
ReagentAcetoneFisher ScientificCatalog #A18P-4
ReagentDAPI (4,6-diamidino-2-phenylindole, dihydrochloride)Thermo Fisher ScientificCatalog #D1306
ReagentHBSSGibco - Thermo Fisher ScientificCatalog #14170-112
ReagentSterile deionized H2O
ReagentProlong Gold Antifade MountantThermo Fisher ScientificCatalog #P36930
ReagentMitoTracker Green FMCell Signaling TechnologyCatalog #9047S
Troubleshooting
Seed fibroblasts in 24 well plates on cover slips at 5,000 cells per well
Culture cells in ReagentDMEM and Hams F-12 50/50Fisher ScientificCatalog #MT10090CV supplemented with 10% ReagentFBS (HyClone) and 1% ReagentAntibiotic/Antimycotic (100X)Thermo Fisher ScientificCatalog #15240062 at Temperature37 °C under 3% O2 and 5% CO2 in ReagentTissue culture dish (10 cm)Merck MilliporeSigma (Sigma-Aldrich)Catalog #Z707651 for Duration48:00:00

Remove media
Wash cells with warm ReagentHBSSThermo Fisher Scientific
MitoTracker Red
Follow this procedure for MitoTracker Red staining and fixed cell imaging
Add Concentration0.3 μL/mL ReagentMitoTracker® Red CMXRosCell Signaling TechnologyCatalog #9082 in FBS-free DMEM for Duration00:30:00

Wash cells twice with Reagent1X PBS

Fix for Duration00:15:00 in the dark with warmReagent4% Formaldehyde, Methanol-FreeCell Signaling TechnologyCatalog #47746

Remove formaldehyde and wash cells twice with Reagent1X PBS for Duration00:05:00

Add Amount500 µL chilled ReagentAcetoneFisher ScientificCatalog #A18P-4 to each well and incubate for Duration00:05:00

Remove acetone and wash cells once with Reagent1X PBS
Add ReagentDAPI (4,6-diamidino-2-phenylindole, dihydrochloride)Thermo Fisher ScientificCatalog #D1306 diluted 1:2,000 in ReagentHBSSGibco - Thermo Fisher ScientificCatalog #14170-112 to each well and incubate for Duration00:15:00

Remove DAPI solution and wash cells with ReagentSterile deionized H2O

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

MitoTracker Green
Follow this procedure for MitoTracker Green staining and live cell imaging
Prepare mix by adding Amount2 µL of ReagentMitoTracker Green FMCell Signaling TechnologyCatalog #9047S to Amount12 mL and warming

Incubate cells in staining mix in FBS-free DMEM for Duration00:30:00

Wash cells once with ReagentHBSSGibco - 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.