Glucocerebrosidase (GCase) activity assay

GCase Activity Assay Using PFB-FDGlu in SH-SY5Y Cells (96-Well Plate Format)

Priya Thakur¹, Han Seok Ko¹

¹Neuroregeneration and Stem Cell Programs, Institute for Cell Engineering, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA; Department of Neurology, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA.

Principle

PFB-FDGlu is a cell-permeable fluorogenic substrate that is hydrolyzed by lysosomal GCase in a live cell to release a fluorescent product. Fluorescence intensity is directly proportional to GCase activity.

Materials

• SH-SY5Y cells 

• PFB-FDGlu substrate 

• Conduritol B epoxide (CBE) 

• DMEM/F12 medium supplemented with 10% FBS 

• PBS (1×) 

• Assay buffer (100 mM citrate-phosphate buffer, pH 5.5) 

• Triton X-100 

• White or black 96-well plates (black plates preferred) 

• Fluorescence plate reader 

Reagent Preparation

1. Assay buffer

Prepare:

• 100 mM citrate-phosphate buffer (pH 5.5) 

• 0.1% Triton X-100 

Store at 4°C.

2. PFB-FDGlu working solution

Prepare a 100 mM stock solution in DMSO.

Dilute in assay buffer to obtain a final concentration of 100 μM.

3. CBE stock

Prepare a 50 mM stock solution in sterile water.

Final concentration in cells: 50 μM.

Cell Seeding

1. Seed SH-SY5Y cells in a black 96-well plate. 

2. Seed 20,000–25,000 cells per well in 100 μL complete medium. 

3. Incubate overnight at 37°C with 5% CO₂. 

Experimental Setup

Group                                Treatment

Blank                                  Medium only

Control                               Vehicle-treated cells

Negative conrtol                 50 μM CBE 

Test compounds                 Desired concentration

Use triplicate wells for each condition.

CBE Treatment

1. Add CBE to the negative-control wells to a final concentration of 50 μM. 

2. Incubate for 24 hours at 37°C. 

3. Add vehicle to control wells. 

GCase Activity Assay

Day 2

1. Remove culture medium. 

2. Wash cells once with 100 μL PBS. 

3. Add 50 μL assay buffer to each well. 

4. Add 50 μL of PFB-FDGlu solution (2× stock) to achieve a final concentration of 100 μM. 

5. Final assay volume: 100 μL per well. 

6. Incubate for 30–60 minutes at 37°C, protected from light. 

Fluorescence Measurement

Measure fluorescence using a plate reader:

• Excitation: 492 nm 

• Emission: 526 nm 

Read fluorescence from the bottom of the plate if possible.

Data Analysis

Background correction

Subtract the fluorescence of blank wells:

Corrected fluorescence = Sample fluorescence − Blank fluorescence

Relative GCase activity

Relative GCase activity = (Sample fluorescence/Control fluorescence) × 100

 

Percentage inhibition by CBE

Inhibition = (1-CBE fluorescence/Control fluorescence)×100
 

Notes

• Use black plates to minimize fluorescence cross-talk. 

• Protect PFB-FDGlu from light. 

• Optimize substrate concentration and incubation time if needed. 

• Include at least three biological replicates.