Jan 11, 2026

Public workspaceProtocol for Lysosomal Hexose Enrichment in SH-SY5Y Cells

DOI
https://dx.doi.org/10.17504/protocols.io.yxmvmb7p5g3p/v1
  • Ali Ghoochani1,2,3,4,
  • Monther Abu-Remaileh1,2,3,4
  • 1Department of Chemical Engineering, Stanford University, Stanford, CA 94305, USA;
  • 2Department of Genetics, Stanford University, Stanford, CA 94305, USA;
  • 3The Institute for Chemistry, Engineering and Medicine for Human Health (Sarafan ChEM-H), Stanford University, Stanford, CA 94305, USA;
  • 4Aligning Science Across Parkinson’s (ASAP) Collaborative Research Network, Chevy Chase, MD, 20815, USA
  • asap
Ali Ghoochani
Icon indicating open access to content
QR code linking to this content
DOI: https://dx.doi.org/10.17504/protocols.io.yxmvmb7p5g3p/v1
Protocol Citation: Ali Ghoochani, Monther Abu-Remaileh 2026. Protocol for Lysosomal Hexose Enrichment in SH-SY5Y Cells. protocols.io https://dx.doi.org/10.17504/protocols.io.yxmvmb7p5g3p/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: July 09, 2025
Last Modified: January 11, 2026
Protocol Integer ID: 222012
Keywords: protocol for lysosomal hexose enrichment, lysosomal hexose enrichment, lysosomes of sh, sy5y cell, sy5y cells this protocol, lysosome, quantification of hexose, enzymatic hydrolysis, hexose, cell, using invertase
Abstract
This protocol outlines the enrichment and quantification of hexoses within lysosomes of SH-SY5Y cells. Cells are subjected to serum starvation and sucrose loading, followed by enzymatic hydrolysis using invertase.
Materials

·       SH-SY5Y cells
·       15 cm cell culture dishes
·       DMEM (with and without serum)
·       Sucrose (Sigma)
·       Invertase from Saccharomyces cerevisiae (Sigma)
·       1x DPBS
Troubleshooting
Procedure
3d
 Seed 15 × 10⁶ SH-SY5Y cells into 15 cm cell culture dishes.
 Incubate cells for 3 days under fed conditions (with serum).

3d
Wash cells twice with 1XDPBS
 Replace medium with serum-free DMEM and incubate cells for 72 hours to induce serum starvation.

Note
This is a general protocol for hexose enrichment of lysosomes . It can be applied to a variety of cell types depending on the specific experimental 

  At 48 hours of serum starvation, supplement the medium with sucrose to a final concentration of 50 mM.

 Continue incubation for an additional 24 hours (total of 72 hours under serum starvation).

After treatment, wash cells thoroughly with 1x DPBS to remove residual sucrose.

 Incubate cells with 0.5 mg/ml invertase in serum-free DMEM for 1 hour at 37°C.

LysoIP
After invertase treatment, proceed with lysosomal isolation using the LysoIP protocol to isolate intact lysosomes.

Find protocol here: dx.doi.org/10.17504/protocols.io.bybjpskn


Note
In the final step of the LysoIP protocol, after three washes, proceed directly with hexose derivatization

Benzoyl-hexose derivatization
 Perform benzoyl-hexose derivatization on the isolated lysosomal fractions for hexose analysis.
Find protocol here: dx.doi.org/10.17504/protocols.io.36wgqnxeygk5/v
Data acquisition and analysis of derivatized hexose/glucose
Find protocol here:
dx.doi.org/10.17504/protocols.io.81wgbw1xygpk/v1