PFB-FDGlu Assay for Measuring Glucocerebrosidase Activity in Human Monocytes
This protocol is adapted from research data and provided for reference only. Adjustments may be necessary based on specific experimental requirements.
(1) Reagent Preparation: Dissolve PFB-FDGlu in dimethyl sulfoxide (DMSO) to prepare a 37.5mmol/L stock solution (5mg PFB-FDGlu dissolved in 154µl DMSO), aliquot and store at -20°C, recommended for use within one week; dissolve CBE (Conduritol B-epoxide) in DMSO to prepare a 50mM stock solution (5mg CBE dissolved in 616.9µl DMSO), aliquot and store at -20°C; prepare FACS buffer containing 5% fetal bovine serum (FBS), 1mM EDTA, and 25mM HEPES, filter-sterilize and store at 4°C; use RPMI 1640 medium (containing 10% FBS, 1% penicillin-streptomycin, and 1% L-glutamine) as the cell culture medium.
(2) Cell Isolation and Preparation: Collect human peripheral blood into BD CPT anticoagulant tubes, centrifuge at 1800×g for 20 minutes at room temperature, and collect the peripheral blood mononuclear cell (PBMC) layer. Wash the cells once with DPBS (300×g, 15 minutes), resuspend in pre-warmed culture medium, perform cell counting, adjust the cell concentration to 1×10⁶ cells per tube, and aliquot into three 1.5ml centrifuge tubes (CBE inhibition group, DMSO control group, and isotype control group, respectively).
(3) Enzyme Inhibition and Substrate Incubation: Add 2µl of 50mM CBE stock solution (final concentration 1mM) to the CBE inhibition tube, add 2µl of DMSO to the DMSO control group, vortex gently, and incubate at 37°C in a 5% CO₂ incubator for 60 minutes. Subsequently, add 2µl of 37.5mM PFB-FDGlu stock solution (final concentration 0.75mM) to both the CBE inhibition tube and the DMSO control group, vortex gently, and continue incubation at 37°C in a 5% CO₂ incubator for 30 minutes.
(4) Reaction Termination and Surface Marker Staining: After incubation, immediately add 1ml of ice-cold FACS buffer to terminate the reaction, centrifuge at 300×g for 5 minutes at 4°C, and discard the supernatant. Resuspend the cells in 95µl of ice-cold FACS buffer, add 5µl of anti-human CD14-PE-Cy7 antibody (for the CBE inhibition tube and DMSO control group) or isotype control antibody (for the isotype control tube), vortex gently, and incubate at 4°C in the dark for 20 minutes. Add 1ml of FACS buffer to wash the cells, centrifuge and discard the supernatant, resuspend the cells in 350µl of FACS buffer, filter through a cell strainer into flow cytometry-specific tubes, and store at 4°C protected from light until testing.
(5) Flow Cytometry Detection and Data Analysis: Use a flow cytometer equipped with a 488nm laser and a 530nm-FITC filter (FL1 channel) to acquire data.
Precautions:
(1) This method is suitable for both freshly isolated and thawed PBMCs (frozen cells require approximately 2 hours of recovery culture after thawing).
(2) The performance of reconstituted PFB-FDGlu will decline with prolonged storage; it is recommended to use it within one week.
(3) For your safety and health, please wear a lab coat and disposable gloves during operation.
References:
[1] Hughes LP, Halliday GM, Dzamko N. Flow Cytometry Measurement of Glucocerebrosidase Activity in Human Monocytes. Bio Protoc. 2020 Apr 5;10(7):e3572. |
