Mar 24, 2025

Public workspaceTransient Transfection of Cultured Mammalian Cells for Live 3D Fluorescence Microscopy

DOI
dx.doi.org/10.17504/protocols.io.ewov1dr82vr2/v1
  • 1UC Berkeley
Samantha C Lewis
Icon indicating open access to content
QR code linking to this content
DOI: dx.doi.org/10.17504/protocols.io.ewov1dr82vr2/v1
Protocol Citation: Eve V. Kakudji, Samantha C Lewis 2025. Transient Transfection of Cultured Mammalian Cells for Live 3D Fluorescence Microscopy. protocols.io https://dx.doi.org/10.17504/protocols.io.ewov1dr82vr2/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: February 20, 2025
Last Modified: March 24, 2025
Protocol Integer ID: 123321
Keywords: Transfection, Fluorescence microscopy, Plasmids, mitochondria, live imaging
Funders Acknowledgements:
Michael J. Fox Foundation
Grant ID: 059455
Abstract
This is a protocol for transient transfection of plasmids encoding tagged mitochondrial proteins into cultured mammalian cells. The result is a sample of live cells in which various features of mitochondrial morphometrics can be observed by fluorescence microscopy.
Guidelines
Timeline

  • D0: Seed U2OS cells (typically at ~ 20-30% confluency, so that they are at ~ 80% confluency on the day of the experiment).
  • D1: Perform transfection – Replace with fresh media 5 hours post-transfection.
  • D2: Perform experiment 24 hours post-transfection.

Materials
Reagents:

  • Complete DMEM media
  1. 500mL ReagentDMEM high glucose no glutamineThermo Fisher ScientificCatalog #11960044
  2. 55mL ReagentFetal Bovine Serum, heat inactivated, qualified, One Shot™, United StatesThermo FisherCatalog #A3840101
  3. 5.5 mL ReagentL-Glutamine (200 mM)Thermo FisherCatalog #25030081
  4. 5.5mL ReagentPenicillin-StreptomycinGibco - Thermo FisherCatalog #15140122

  • ReagentOpti-MEM™ I Reduced Serum MediumThermo Fisher ScientificCatalog #31985070
  • ReagentLipofectamine™ 2000 Transfection ReagentThermo Fisher ScientificCatalog #11668030
  • Plasmids
  1. ReagentmCherry-TOMM20-N-10addgeneCatalog #55146
  2. ReagentATP5F1B (NM_001686) Human Tagged ORF CloneOriGeneCatalog #RG201638

  • 35-nm glass bottom dishes (Mattek, P35GC-1.5-14-C)
  • Pipettes
  • Pipette tips
  • 1.5mL or 2mL microtubes

Equipment:

  • Incubator
  • Biosafety cabinet
  • Fluorescence microscope







Procedure
Procedure
1d 5h 15m
1d 5h 15m
Seed U2OS cells in DMEM complete medium onto 35-nm glass bottom imaging dishes.

Incubate the cells at Temperature37 °C , 5% CO2 for ~Duration24:00:00 .

1d
Incubation
Thaw plasmid(s) and measure your desired concentration for transfection (Amount500 ng for mCherry-TOMM20-N-10 and Amount250 ng for ATP5F1B-tGFP).

Take an aliquot of Opti-MEM and let it warm at TemperatureRoom temperature in the dark.

Take a microfuge tube: add Amount100 µL of Opti-MEM and Amount4 µL of lipofectamine 2000 per transfection (example: if you are doing two transfections, you will need Amount200 µL Opti-MEM and Amount8 µL lipofectamine 2000). Mix gently.

Pipetting
Mix
Take microfuge tubes. In each tube, add Amount100 µL of Opti-MEM and your desired concentration of plasmid (example: Amount500 ng for mCherry-TOMM20-N-10 /Amount250 ng for ATP5F1B-GFP). Mix gently.

Pipetting
Mix
Add Amount104 µL of the Opti-MEM/Lipofectamine 2000 mix in each of the tubes prepared in step 6. Mix gently.

Pipetting
Mix
Let the tubes incubate at TemperatureRoom temperature for Duration00:15:00 .

15m
Incubation
Take the cells out of the incubator and add the transfection mixture dropwise to cells.

Pipetting
Incubate the cells at Temperature37 °C , 5% CO2 for Duration05:00:00 .

5h
Incubation
Replace media with fresh DMEM complete media.

Perform experiment 24 hours post-transfection.

Acknowledgements
We thank the Lewis Lab for helpful discussion.