Dec 17, 2024

Public workspaceFatty Acid Induction to Hippocampal Neurons

DOI
dx.doi.org/10.17504/protocols.io.n2bvj93jxlk5/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.n2bvj93jxlk5/v1
Protocol Citation: Mukesh Kumar, Timothy A. Ryan 2024. Fatty Acid Induction to Hippocampal Neurons. protocols.io https://dx.doi.org/10.17504/protocols.io.n2bvj93jxlk5/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 17, 2024
Protocol Integer ID: 114387
Keywords: ASAPCRN
Funders Acknowledgements:
NIH
Grant ID: NS036942
NIH
Grant ID: NS11739
ASAP
Grant ID: ASAP-024404
Abstract
This protocol provides a stepwise procedure for preparing homogeneous fatty acid–BSA complexes for lipid droplet induction in primary cultured hippocampal neurons. The protocol also describes the fatty acid induction to primary hippocampal neurons for electrical stimulation experiments.
Guidelines
The protocol needs prior approval by the users' Institutional Animal Care and Use Committee (IACUC) or equivalent ethics committee.
Materials
Materials:

  • ReagentOleic acidMerck MilliporeSigma (Sigma-Aldrich)Catalog #O1383
  • ReagentPalmitic acidMerck MilliporeSigma (Sigma-Aldrich)Catalog #P0500
  • ReagentBSAMerck MilliporeSigma (Sigma-Aldrich)Catalog ##A8806
  • Tris Buffer: 0.1 M Tris (pH 7.4 using NaOH)
  • NaOH Solution: 0.1 M NaOH (200 mg NaOH in 50 ml ultrapure water)
  • NaCl Solution: 0.9% NaCl (450 mg NaCl in 50 ml ultrapure water)
  • ReagentBODIPY™ 493/503 (4,4-Difluoro-1,3,5,7,8-Pentamethyl-4-Bora-3a,4a-Diaza-s-Indacene)Thermo FisherCatalog #D3922

Equipments:

  • Heating block (temperature controlled to 40°C and 70°C as required)
  • Syringe filter: 0.22 μm pore size
  • Culture hood
  • Aluminum foil
  • Ultrapure water
  • Vortex heater



Procedure to prepare Oleic Acid–BSA (OA-BSA) Complex
Procedure to prepare Oleic Acid–BSA (OA-BSA) Complex
1d 3h 10m
1d 3h 10m
Prepare 0.1 M Tris Buffer (pH 7.4):

Dissolve Trizma in ultrapure water and adjust pH to 7.4 using NaOH.

Dissolve BSA:
Pour Amount4 mL of Concentration0.1 Molarity (M) Tris into a 50 ml tube. Overlay Amount697.5 mg of FA-free BSA on top.
Pipetting
Place the tube on a heating block maintained at Temperature42 °C and stir gently until the BSA is completely dissolved. Adjust the final volume to Amount4.98 mL with Concentration0.1 Molarity (M) Tris buffer.
Pipetting
Mix
Add Oleic Acid:

Thaw OA at Temperature37 °C . Add Amount19.83 µL of OA to the BSA solution.

Pipetting
Incubate:

Wrap the tube with aluminum foil to protect from light. Incubate at Temperature37 °C with gentle shaking for Duration02:00:00 -Duration03:00:00 . Ensure that the solution becomes homogeneously clear (turbidity should disappear).

3h
Incubation
Filter the Complex:

Filter the solution through a 0.22 μm syringe filter in a sterile culture hood. Aliquot Amount250 µL portions and store at Temperature4 °C .

Induction of Lipid droplets:

Add OA-BSA complex to 14-21 DIV culture primary hippocampal neurons/fibroblasts to the final concentration of Amount200 µL -Amount400 µL for Duration24:00:00 .

1d
Pipetting
Detection of lipid droplets:

Add Amount1 µL BODIPY to the culture media and return the dishes to incubator for Duration00:10:00 . Wash the cells with PBS and mount the glass coverslip to image on confocal microscope.

10m
Incubation
Pipetting
Wash
Procedure to prepare Palmitic Acid–BSA (PA-BSA) Complex
Procedure to prepare Palmitic Acid–BSA (PA-BSA) Complex
4h 20m
4h 20m
Prepare Solutions:

Prepare Concentration0.1 Molarity (M) NaOH by dissolving Amount200 mg NaOH in Amount50 mL ultrapure water.
Pipetting
Prepare 0.9% NaCl Solution by dissolving Amount450 mg NaCl in Amount50 mL ultrapure water.
Pipetting
Dissolve BSA:
Pour Amount3.5 mL of 0.9% NaCl solution into a 50 ml tube. Overlay Amount555.55 mg of FA-free BSA on top.
Place the tube on a heating block maintained at Temperature40 °C and stir gently until the BSA is completely dissolved.
Adjust the final volume to Amount4 mL with 0.9% NaCl solution. Aliquot Amount800 µL portions into 5 Eppendorf tubes and maintain at Temperature42 °C on a vortex heater.
Dissolve Palmitic Acid:

Weigh Amount15.38 mg of PA and dissolve in Amount1 mL of Concentration0.1 Molarity (M) NaOH. Heat the mixture to Temperature70 °C on a heating block, carefully monitoring the temperature to prevent overheating.

Complexing PA with BSA:

Immediately add Amount200 µL of the PA solution to each Amount800 µL aliquot of BSA maintained at Temperature40 °C while vortexing.

Note
PA may solidify if the temperature drops.

Pipetting
Incubate:

Leave PA-BSA solution under agitation for Duration00:15:00 -Duration00:20:00 . The solution may appear turbid/whitish at this stage.

20m
Incubation
Filter the Complex:

Filter the solution through a 0.22 μm syringe filter in a sterile culture hood. Prepare the complex fresh before use and maintain the temperature at Temperature37 °C until used.

Induction of neurons:

Add PA-BSA complex to the media of primary hippocampal neurons to the final concentration of Concentration150 micromolar (µM) for Duration04:00:00 .

Note
  • Protect fatty acid solutions from light during incubation to prevent photooxidation.
  • Ensure precise temperature control during the preparation of both complexes to avoid solidification or denaturation.
  • Always use sterile techniques and equipment to prevent contamination.

4h
Pipetting