Jul 30, 2023
Measurement of GCase activity in lysosomes in live cells
This protocol is a draft, published without a DOI.
- 1UCL
Protocol Citation: rachel.bates Bates 2023. Measurement of GCase activity in lysosomes in live cells. protocols.io https://protocols.io/view/measurement-of-gcase-activity-in-lysosomes-in-live-cxwzxpf6
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 30, 2023
Last Modified: May 31, 2024
Protocol Integer ID: 85689
Keywords: ASAPCRN
Abstract
Assay uses the substrate PFB-FDGluc (5-(Pentafluorobenzoylamino)Fluorescein Di-β-D-Glucopyranoside)from
ThermoScientific (Cat# P11947). Substrate is supposed to be taken up by late
endosomes and lysosomes only and fluoresces green when cleaved by lysosomal GCase.
Described by Mazzulli et al., J
Neurosci. 2016 Jul
20;36(29):7693-706.
Materials
PFB-FDGlu (5-(Pentafluorobenzoylamino)Fluorescein Di-β-D-Glucopyranoside)
Catalog number: P11947
Opti-MEM™ I Reduced Serum Medium
Catalog number: 31985062
Resuspend 5mg of substrate in 100 ul methanol to get 50mg/mL stock. Do not expose to light. Store at -20C
Culture cells in 48 well plate. Should be over 70% confluent to ensure a robust signal. If too confluent activity will plateau rapidly.
1d
For control wells add 10uM CBE or 100uM Bafilomycin overnight.
Wash cells once carefully with prewarmed PBS.
5m
Working substrate solution is 400 mg/ml (I don’t get signal with lower
concentrations). Prepare in OPTIMEM prewarmed to 37 °C: 125 ml/well.
Therefore 1 ul of 50mg/ml stock substrate per 125 ml.
Add 125ul substrate (400ug/ml substrate (400 mg/ml in OPTIMEM) per well. Put in 37 °C incubator for 1 h.
1h
Aspirate substrate. Wash cells three times with 250 ml PBS (37 °C).
10m
ADD 125UL ml OPTIMEM (37 °C). Measure t=0 minutes on plate reader: Ex, 488nm, Em, 520 nm.
Return cells to incubator.
Measure fluorescence every 20-30 minutes in green channel (excitation/emission maxima ∼492/516 nm) for up to 3 hours. Keep checking cells are alive with microscope.
3h
Optional: At end of experiment carefully aspirate OPTIMEM. Lyse cells in wells with 200 ml 1% TX-100 in PBS on ice for 15 minutes. Pellet debris at 17,000 xg, 10 minutes, 4°C. Measure protein concentration in supernatant with BCA assay.