Aug 04, 2024

Public workspaceEndocytosis and internalization assay in primary neuronal culture

DOI
dx.doi.org/10.17504/protocols.io.4r3l22j5ql1y/v1
  • 1Duke Univeristy;
  • 2Duke University
  • West lab protocols
Arpine Sokratian
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DOI: dx.doi.org/10.17504/protocols.io.4r3l22j5ql1y/v1
Protocol Citation: Arpine Sokratian, andrew.west west 2024. Endocytosis and internalization assay in primary neuronal culture. protocols.io https://dx.doi.org/10.17504/protocols.io.4r3l22j5ql1y/v1
Manuscript citation:
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: January 22, 2024
Last Modified: August 05, 2024
Protocol Integer ID: 93919
Keywords: ASAPCRN
Funders Acknowledgements:
ASAP
Grant ID: ASAP-020527
Abstract
This protocol outlines a method for quantifying endocytosis in primary neuronal cultures, specifically focusing on different a-synuclein fibril structures. By utilizing pH-sensitive dye conjugated to experimental proteins/fibrils, we can identify endocytosis pathways and analyze intensity changes in the pHrodo-specific channel from real-time acquired images at various time points.
Materials
ReagentEIPAcaymanchemCatalog #14406
ReagentDyngoAbcamCatalog #ab120689
ReagentMethyl-β-cyclodextrin Merck MilliporeSigma (Sigma-Aldrich)Catalog #C4555 ReagentPitstop® 2AbcamCatalog #ab120687 ReagentWortmanninMerck MilliporeSigma (Sigma-Aldrich)Catalog #W1628-1MG

Protocol materials
ReagentEIPAcaymanchemCatalog #14406
ReagentDyngoAbcamCatalog #ab120689
ReagentMethyl-β-cyclodextrin Merck MilliporeSigma (Sigma-Aldrich)Catalog #C4555
ReagentPitstop® 2AbcamCatalog #ab120687
ReagentWortmanninMerck MilliporeSigma (Sigma-Aldrich)Catalog #W1628-1MG
ReagentHoechst 33342Catalog #H3570
ReagentLysoTracker™ Green DND-26 - Special PackagingThermo FisherCatalog #L7526
ReagentpHrodo™ Red Dextran, 10,000 MW, for EndocytosisThermo FisherCatalog #P10361
Preparation of endocytosis inhibitors
Preparation of endocytosis inhibitors
For the assay we use DIV7 primary hippocampal neuron culture plated in 48-well plates
Prepare stock solution of the endocytosis inhibitors in DMSO:
AB
2.3 mMWortmannin
50 mMDyngo
10 mMPitstop 2
384 mMmethyl-β-cyclodextrin (MβCD)
50 mMethyl-isopropyl amiloride
Recommended concentrations for stock solutions (stocks can be freezed at -80C for couple of days

At the day of the experiment, thaw down the stock solutions in a water bath and dilute the drugs to reach concentrations:



ABCDEFG
Ethyl-isopropyl amiloride (EIPA)50 uM500uM: 10 ul of stock + 990ul PBS
Dyngo10 uM10 mM: 20ul of the drug + 80 ul (50% DMSO)for 100 uM of Dyngo: 5 ul of the 10mM dyngo + 495 ul of PBS
Wortmannin0.2 uM10 uM: 4ul of the stock + 916 ul PBS1uM: 200 ul of 10 uM + 800 ul of PBS
Pitstop 215 uM150 uM: 15ul of P + 984 ul PBS
Methyl-β-cyclodextrin (MβCD) 2 mM20 mM: 25 ul of stock + 475 ul PBS
Drugs are prepared to be diluted to final concentration in the cell culture

Before adding protein/drug of interest, add endocytosis inhibitors 30 minutes before the treatment. As calculated for 48-well plates with 3 mL of the media, 30 ul of diluted drugs are sufficient. ** Note: Be careful adding the diluted drugs (should be RT). Place the plate back to the cell culture incubator.
After 30 minutes, add the protein/drug of interest supplement with ReagentHoechst 33342Contributed by usersCatalog #H3570
As for phRodo-conjugated fibrils, please, see the protocol describing the conjugation process (step 5):
As a control for endocytosis add lysotrackerReagentLysoTracker™ Green DND-26 - Special PackagingThermo FisherCatalog #L7526 and phrodo-10kDA dextran ReagentpHrodo™ Red Dextran, 10,000 MW, for EndocytosisThermo FisherCatalog #P10361

Protocol
Preparation of mouse and human α-synuclein fibrils
NAME
Preparation of mouse and human α-synuclein fibrils
CREATED BY
Arpine Sokratian




After two hours of incubation, start taking images of each well at the high-speed (high sensitive mode)
Continue acquiring images every two to four hours up to 48 hours of incubation.
Calculate the images using cell count (Hoechst) for normalization and calculate the signal for each time-point as a proportion to the maxima of the signal

Calculate the signal from Lysotracker after 30 minutes of incubation and compare the control (no endocytosis inhibitors) to the experimental reactions.