Dec 16, 2024

Public workspaceBODIPY 558/568 C12 (Red-C12) Tracing in Hippocampal Neurons

DOI
dx.doi.org/10.17504/protocols.io.dm6gp9328vzp/v1
  • Mukesh Kumar1,
  • Timothy A. Ryan1,2
  • 1Department of Biochemistry, Weill Cornell Medicine, New York, NY 10065;
  • 2Aligning Science Across Parkinson's (ASAP) Collaborative Research Network, Chevy Chase, Maryland 20815, USA
Mukesh Kumar
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DOI: dx.doi.org/10.17504/protocols.io.dm6gp9328vzp/v1
Protocol Citation: Mukesh Kumar, Timothy A. Ryan 2024. BODIPY 558/568 C12 (Red-C12) Tracing in Hippocampal Neurons. protocols.io https://dx.doi.org/10.17504/protocols.io.dm6gp9328vzp/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: November 27, 2024
Last Modified: December 16, 2024
Protocol Integer ID: 114385
Keywords: ASAPCRN
Funders Acknowledgements:
NIH
Grant ID: NS036942
NIH
Grant ID: NS11739
ASAP
Grant ID: ASAP-024404
Abstract
This protocol provides a detailed method for tracing BODIPY 558/568 labelled fatty acid (Red-C12) from lipid droplets to mitochondria in hippocampal neurons.
Guidelines
The protocol needs prior approval by the users' Institutional Animal Care and Use Committee (IACUC) or equivalent ethics committee.
Materials
Materials:

  • Hippocampal neurons (Isolated from neonatal P0-1 rat pups and cultured sparsely)
  • ReagentBODIPY 558/568 C12Cayman Chemical CompanyCatalog #27014
  • ReagentKLH45Merck MilliporeSigma (Sigma-Aldrich)Catalog #SML1998
  • HBSS (Hank’s Balanced Salt Solution)
  • ReagentEtomoxirMerck MilliporeSigma (Sigma-Aldrich)Catalog #E1905
  • TTX (Tetrodotoxin) (Abcam, ab120055)
  • ReagentMitoTracker™ Green FM - Special PackagingThermo FisherCatalog #M7514

Equipments:

  • Tissue culture incubator
  • Heating block
  • Confocal microscope: Zeiss 880




Red-C12 Pulse Treatment
Red-C12 Pulse Treatment
1d
1d
Add Concentration5 micromolar (µM) KLH45 and Concentration10 micromolar (µM) BODIPY 558/568 C12 (Red-C12) to the feeding media.

Transfer cover slips with sparsely cultured hippocampal neurons to the prepared media and incubate for Duration24:00:00 in a tissue culture incubator at Temperature37 °C with 5% CO2.

1d
Incubation
Incorporation Period
Incorporation Period
1h
1h
After 24 hours, transfer the neurons to complete feeding media without Red-C12.

Incubate for Duration01:00:00 to allow complete incorporation of Red-C12 into neuronal lipid droplets (LDs).

1h
Incubation
Wash and Chase
Wash and Chase
Wash the neurons gently with HBSS to remove excess media and non-incorporated Red-C12.

Wash
Transfer the neurons to fresh HBSS.

Chase the neurons for the defined period (0 to 4.5 hours) under the following conditions:

Control: HBSS alone
Etomoxir Treatment: Concentration10 micromolar (µM) Etomoxir in HBSS

TTX Treatment: Concentration5 micromolar (µM) TTX in HBSS

MitoTracker Staining
MitoTracker Staining
30m
30m
At 30 minutes before the end of the chase period, add Concentration50 nanomolar (nM) MitoTracker Green FM to the media.

Return the neurons to the incubator for Duration00:30:00 .

30m
Incubation
Final Wash
Final Wash
Wash the neurons once with HBSS to remove excess dye.

Wash
Transfer the neurons to fresh HBSS (containing the same inhibitors as in the chase condition, if applicable) preconditioned in the incubator.

Imaging Preparation
Imaging Preparation
Mount the coverslip containing neurons onto a custom-designed imaging chamber.

Ensure the chamber is appropriately sealed and filled with fresh HBSS to maintain neuronal viability.

Imaging and Analysis
Imaging and Analysis
Place the imaging chamber on the stage of a confocal microscope.

Use appropriate settings to image:

Red-C12: Excitation/emission at ~558/568 nm.

MitoTracker Green FM: Excitation/emission at ~490/516 nm.

Capture images at the desired time points and fields of view.

Select distinctly isolated mitochondria on axons and capture fluorescence intensity of Red-C12.

Note
  • Protect Red-C12 and MitoTracker from light to prevent photobleaching.
  • Maintain consistent temperature and conditions during imaging to ensure neuronal viability.
  • Ensure inhibitors (Etomoxir and TTX) are freshly prepared.
  • Avoid analysis of the mitochondria from somatic and dendritic regions of neurons.