Dec 16, 2024

Public workspaceIsolation of Lipid Droplets from Cultured Cortical Neurons

DOI
dx.doi.org/10.17504/protocols.io.n2bvj934plk5/v1
  • Mukesh Kumar1,
  • Daehun Park2,3,4,
  • Pietro De Camilli2,3,4,
  • 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;
  • 3Department of Neuroscience, Howard Hughes Medical Institute, Program in Cellular Neuroscience, Neurodegeneration and Repair, Yale University School of Medicine, New Haven, Connecticut 06520, USA;
  • 4Department of Cell Biology, Yale University School of Medicine, New Haven, Connecticut 06520, USA
Mukesh Kumar
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DOI: dx.doi.org/10.17504/protocols.io.n2bvj934plk5/v1
Protocol Citation: Mukesh Kumar, Daehun Park, Pietro De Camilli, Timothy A. Ryan 2024. Isolation of Lipid Droplets from Cultured Cortical Neurons. protocols.io https://dx.doi.org/10.17504/protocols.io.n2bvj934plk5/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 26, 2024
Last Modified: December 16, 2024
Protocol Integer ID: 114539
Keywords: ASAPCRN
Funders Acknowledgements:
NIH
Grant ID: NS036942
NIH
Grant ID: NS11739
ASAP
Grant ID: ASAP-024404
Abstract
This protocol provides a robust method for purifying lipid droplets from cultured cortical neurons, suitable for lipidomic analysis of constituent lipids.
Guidelines
The protocol needs prior approval by the users' Institutional Animal Care and Use Committee (IACUC) or equivalent ethics committee.
Materials
  • Cortical neurons: 8-10 × 15 cm dishes (~80% confluent) 14-21 DIV
ReagentKLH45Merck MilliporeSigma (Sigma-Aldrich)Catalog #SML1998
  • MEPS Buffer Components:

  1. ReagentPIPESMerck MilliporeSigma (Sigma-Aldrich)Catalog #P6757
  2. ReagentEGTAMerck MilliporeSigma (Sigma-Aldrich)Catalog #E3889
  3. ReagentMagnesium sulfate solutionMerck MilliporeSigma (Sigma-Aldrich)Catalog #M3409
  4. ReagentPotassium hydroxide ACS reagent, ≥85%, pelletsMerck MilliporeSigma (Sigma-Aldrich)Catalog #221473-500G

  • cOmplete Protease Inhibitor Cocktail (Roche, 45-11697498001)
  • ReagentSucroseMerck MilliporeSigma (Sigma-Aldrich)Catalog #S0389
  • ReagentBODIPY™ 493/503 (4,4-Difluoro-1,3,5,7,8-Pentamethyl-4-Bora-3a,4a-Diaza-s-Indacene)Thermo FisherCatalog #D3922
  • ReagentCellMask™ Orange Plasma membrane StainThermo FisherCatalog #C10045

Equipment:

  • Nitrogen Cavitation Apparatus
  • Centrifugation Equipment:
  1. Table-top centrifuge (capacity 2 ml, up to 1800 g),
  2. Ultracentrifuge with swinging-bucket rotor (SW41Ti, Beckman Coulter; up to 1,74,000 g)
  • Needles and Syringes: 25 gauge needles with 2 ml syringes.




Neuronal Preparation
Neuronal Preparation
Isolate cortical neurons from P0-P1 pups and culture them in 15 cm dishes (8-10 plates).
Maintain the neurons in standard culture condition for 14-21 days to allow growth of axons and synapse formation.
Treat the neurons with Concentration5 micromolar (µM) KLH45 (dissolved in DMSO) for 24 hours prior to harvesting.

Pipetting
Harvesting and Lysis
Harvesting and Lysis
Harvest neurons in Amount1.5 mL of Concentration0.9 Molarity (M) MEPS buffer supplemented with 1X protease inhibitor cocktail.

MEPS buffer composition: PIPES: 35 mM (adjust pH to 7.2 with KOH), EGTA: 5 mM, MgSO₄: 5 mM, Sucrose: 0.9 M

Pipetting
Lyse the neurons using the nitrogen cavitation method or Isobiotec Cell Homogenizer (8-10 strokes with 16-micron clearance). Confirm complete lysis under an inverted white-field or phase-contrast microscope.
Post-Nuclear Supernatant (PNS) Preparation
Post-Nuclear Supernatant (PNS) Preparation
10m
10m
Centrifuge the lysate at Centrifigation1800 x g, 4°C, 00:10:00 to remove unlysed somas, nuclei, and plasma membrane fractions.
10m
Centrifigation
Temperature
Collect the supernatant (called post-nuclear supernatant, PNS) into a fresh tube.
Gradient Setup and Ultracentrifugation
Gradient Setup and Ultracentrifugation
1h 30m
1h 30m
Pour the PNS over 2.5 M MEPS in a clear ultracentrifuge tube (SW41).
Overlay the bottom layer with Amount2 mL each of Concentration1.2 Molarity (M) , Concentration0.5 Molarity (M) , and Concentration0 Molarity (M) MEPS to establish a step gradient.

Pipetting
Load the ultracentrifuge tube into the SW41Ti rotor and balance it against an identical tube.
Centrifuge the gradient at Centrifigation174000 x g, 4°C, 01:30:00 .

1h 30m
Centrifigation
Temperature
Lipid Droplet (LD) Collection
Lipid Droplet (LD) Collection
Carefully transfer the ultracentrifuge buckets to ice.
Collect the LDs floating on the top of gradient using a syringe fitted with appropriate needles (25 gauge) or a tube slicer.
Storage
Storage
Snap-freeze the collected LDs in liquid nitrogen for future use.
Validation of Purity
Validation of Purity
Perform western blotting to confirm the purity of the isolated LDs.
Confirm enrichment of LD by Perilipin-2 and contamination (if any) using organelle-specific markers for mitochondria, ER, Golgi, and endosomes.

Note
• Appropriate clearance/number of strokes with Isobiotec cell Homogenizer, or pressure during Nitrogen cavitation is critical for effective lysis while preserving organelle integrity.
• Extreme care is required during the gradient setup to prevent contamination of the LD layer.
• Ensure all ultracentrifuge tubes are properly balanced to avoid damage of rotor/centrifuge.
• Cell-Mask Plasma Membrane Stain can be used to confirm absence of unwanted membrane contamination in LDs samples.