Jul 08, 2023

DQ-BSA assay

DOI
https://dx.doi.org/10.17504/protocols.io.kxygx9r9dg8j/v1
  • Narayana Yadavalli1,2,3,4,5,
  • Shawn M. Ferguson1,2,3,4,6,5
  • 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
Berrak Ugur
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DOI: https://dx.doi.org/10.17504/protocols.io.kxygx9r9dg8j/v1
Protocol Citation: Narayana Yadavalli, Shawn M. Ferguson 2023. DQ-BSA assay. protocols.io https://dx.doi.org/10.17504/protocols.io.kxygx9r9dg8j/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: May 26, 2023
Last Modified: June 01, 2024
Protocol Integer ID: 82508
Keywords: DQ BSA, BSA-488, Lysosomes, ASAPCRN, lysosomal proteolytic activity, dq bsa, assay, dq
Funders Acknowledgements:
ASAP
Grant ID: 000580
Abstract
This protocol describes the DQ BSA assay for measuring the lysosomal proteolytic activity.
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Materials
Reagents

  1. DQ BSA - #D12051 (1mg/ml prepared in sterile PBS), working concentration 10μg/ml.
  2. BSA-488 - #A13100 (5mg/ml prepared in sterile PBS), working concentration 5 μg/ml.
  3. Mattek glass bottom dishes


DQ-BSA assay
3h 10m
Plate 100,000 mature macrophages or microglia or BMDM were seeded on Mattek glass bottom dishes.
Next day treat cells with 50 nanomolar (nM) MLi2 or 250 nanomolar (nM) LRRK2-IN-1 for 02:00:00 followed by 01:00:00 treatment with 10 µg/mL DQ-BSA (Thermo Fisher Scientific, #D12051) and 50 µg/mL Alexa-488 BSA (Thermo Fisher Scientific #A13100) in the media containing inhibitors.

3h
After 3 hours wash the cells 3X with culture media.
Then add 1 mL culture media to the washed cells and return to incubator for 00:10:00 .

10m
Imaging was done on LSM 880.