Dec 03, 2024

Public workspaceSPION-mediated Lysosome Purification – RAW264.7

The Journal of Cell Biology
DOI
dx.doi.org/10.17504/protocols.io.e6nvwbk32vmk/v1
  • 1Department of Cell Biology, Yale University School of Medicine, New Haven, Connecticut 06510, USA;
  • 2Neuroscience, Yale University School of Medicine, New Haven, Connecticut 06510, USA;
  • 3Program in Cellular Neuroscience, Neurodegeneration and Repair;
  • 4Wu Tsai Institute Yale University School of Medicine, New Haven, Connecticut 06510, USA;
  • 5Aligning Science Across Parkinson’s (ASAP) Collaborative Research Network, Chevy Chase, MD, 20815, USA;
  • 6Kavli Institute for Neuroscience, Yale University School of Medicine, New Haven, Connecticut 06510, USA
Amanda Bentley-DeSousa
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DOI: dx.doi.org/10.17504/protocols.io.e6nvwbk32vmk/v1
Protocol Citation: Amanda Bentley-DeSousa, Shawn Ferguson 2024. SPION-mediated Lysosome Purification – RAW264.7. protocols.io https://dx.doi.org/10.17504/protocols.io.e6nvwbk32vmk/v1
Manuscript citation:
https://doi.org/10.1101/2023.10.31.564602
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: December 02, 2024
Last Modified: December 03, 2024
Protocol Integer ID: 113414
Keywords: RAW 264.7, lysosome isolation, SPION
Funders Acknowledgements:
Aligning Science Across Parkinson’s Disease
Grant ID: ASAP-000580
Abstract
This protocol details steps taken to isolate lysosomes in RAW 264.7 cells.
Materials
DMEM (Thermo Fisher Scientific, 11965-092)
FBS (Thermo Fisher Scientific, 16140-071)
Penicillin/Streptomycin (Thermo Fisher Scientific, 15140122)
CellStripper (Corning, 356230)
PBS [1.1 mM KH2PO4 (J.T. Baker, 3246-01), 155.2 mM NaCl (Sigma-Aldrich, 3624-05), and 3 mM Na2HPO4 (J.T. Baker, 3828-05)]
SPION Particles (see https://www.protocols.io/view/synthesis-of-colloidal-dextran-conjugated-superpar-eq2lyn69pvx9/v1)
5 mM HEPES (pH 7.4, Thermo Fisher Scientific, 15630-080)
HB buffer (5 mM Tris Base (American Bio, AB02000-05000), 250 mM Sucrose (Sigma-Aldrich, S0389), and 1 mM EGTA pH 7.4, Sigma-Aldrich, E4378) supplemented with inhibitors [cOmplete mini EDTA-free protease inhibitor (Roche, 11836170001) and PhosSTOP (Roche, 4906837001)]
Dounce Homogenizers (DWK Life Sciences WheatonTM Dounce Tissue grinders, 06-434)
LS columns (Miltenyi Biotec, 130042401)
QuadroMACS separator (Miltenyi Biotec, 130-091-051)
Day 0
Day 0
Split a ~90% confluent 15-cm dish of RAW 264.7 cells into 8 10-cm dishes.
4 10-cm dishes are sufficient for one condition (for example, WT DMSO treatment).
Day 1
Day 1
3h 40m
3h 40m
Replace the media in each dish with DMEM containing FBS and P/S supplemented with 5 mM HEPES and 5% SPION particles for Duration01:00:00 .

1h
Wash each dish 2X with PBS.
Add fresh media (DMEM containing FBS and P/S) to wash out the SPIONs for Duration02:00:00 .

2h
At this point, any drug treatment can be added.
Wash each dish 2X with cold PBS on ice. Scrape each dish with Amount1 mL of PBS and combine each condition into a 15-mL Falcon tube.

For example, all 4 10-cm dishes from WT DMSO condition into 1 Falcon tube.
Centrifuge Centrifigation300 rcf, 4°C, 00:05:00 each 15-mL Falcon tube.

5m
Remove the supernatant and resuspend the pellet in Amount1 mL ice-cold HB buffer for Duration00:05:00 on ice.

5m
HB buffer: 5 mM Tris Base, 250 mM Sucrose, and 1 mM EGTA pH 7.4 supplemented with inhibitors (cOmplete mini EDTA-free protease inhibitor and PhosSTOP).
Transfer the lysate to a Dounce homogenizer and homogenize 50 times each sample with the tight pestle on ice.
Centrifuge Centrifigation800 rcf, 4°C, 00:10:00 the lysate to obtain the cell lysate.

10m
At this step, a portion of the supernatant cell lysate was kept for the “Cell Lysate”.
Wash LC columns once with Amount2.5 mL of HB buffer on a QuadroMACS separator.

Apply the remainder of the supernatant/cell lysate to the LC columns. Collect the flow-through and reapply it to the column.
Wash LC columns with Amount3 mL of HB buffer on a QuadroMACS separator.

Remove LC columns from the QuadroMACS separator and elute lysosomes inao ultracentrifuge tubes with Amount2.5 mL of HB buffer.

Ultracentrifuge Centrifigation55000 rpm, 4°C, 00:20:00 using a TLA-100.3 rotor in a Beckman-Coulter Ultracentrifuge Max Optima.

20m
Remove the supernatant and resuspend the lysosome pellet in Amount50 µL of HB buffer and freeze at -20C.

Day 2+
Day 2+
Quantify protein concentrations using Coomassie Plus Protein Assay Reagent as per the manufacturer’s protocol. Prepare lysates for immunoblotting as in the Protein Lysate and Immunoblotting protocol.io.
Protocol references
This protocol was adapted from Rofe and Pryor, 2016 (DOI: 10.1101/pdb.prot084822).