Feb 10, 2025
Lysosomal and mitochondrial functional assays in human neurons
- 1Stanford University
Protocol Citation: Ching-Chieh Chou, Judith Frydman 2025. Lysosomal and mitochondrial functional assays in human neurons. protocols.io https://dx.doi.org/10.17504/protocols.io.261gerknol47/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: February 10, 2025
Last Modified: February 10, 2025
Protocol Integer ID: 119906
Keywords: ASAPCRN
Funders Acknowledgements:
Aligning Science Across Parkinson's
Grant ID: ASAP-024268
Aligning Science Across Parkinson's
Grant ID: ASAP-000282
Abstract
Cellular assays to test lysosomal and mitochondrial functions in human neurons at basal and stressful conditions.
Lysosomal acidification
Lysosomal acidification
Dextran, Fluorescein, 40,000 MW, Anionic, Lysine Fixable (Thermo Fisher Scientific)
Dextran, Tetramethylrhodamine, 70,000 MW, Lysine Fixable (Thermo Fisher Scientific)
Conjugated Dextrans are reconstituted at stock concentration of 25 mg/ml in 1 ml H2O and stored at -20°C.
Human tNeurons are seeded at 5 × 104 per well of a 24-well plate or 5 ×103 per well of a 96-well plate.
Cells are incubated with conjugated Dextrans at 0.5 mg/mL for 4 hr at 37°C.
Remove medium and rinse with PBS once.
Add fresh culture media and chase for additional 20 hr to accumulate Dextran in late endosomes and lysosomes.
Cells are treated with DMSO or any pharmacological perturbations.
Cells are fixed by 4% PFA for 15 min and prepared for imaging by the confocal microscope or CLARIOstar plate reader.
Lysosomal degradation/proteolysis
Lysosomal degradation/proteolysis
Magic Red Cathepsin-B substrate, MR-(RR)2, (ImmunoChemistry, #938).
Magic Red Cathepsin-B substrate is reconstituted in DMSO and stored at -20°C.
When cells are available for experiments, Magic Red Cathepsin-B substrate is diluted with H2O at
1:10 ratio and added to cell culture medium at a dilution of 1:25 to form 1X staining solution.
Human tNeurons are incubated with Magic Red Cathepsin-B substrate for 30 min at 37°C.
To test toxic effects on lysosomal degradation revealed by Cathepsin-B activity, cells are treated with DMSO or any pharmacological perturbations.
Remove the media and rinse twice with PBS.
Cells are fixed by 4% PFA for 15 min and prepared for imaging by the confocal microscope or CLARIOstar plate reader
Lysosomal calcium
Lysosomal calcium
Cal-520-Dextran Conjugates at 3 kDa (AAT Bioquest).
Cal-520 is reconstituted in DMSO, aliquoted into single-use volumes and stored at -20°C.
Human tNeurons are plated on sterile multi-chamber glass bottom slides for 5 weeks and
incubated with 5 μM Cal-520 and 0.1 μM LysoTracker Red DND-99 (Thermo Fisher
Scientific) for 2 hr at 37°C.
After washing off excess dye by PBS, cells are treated with DMSO or any pharmacological perturbations.
Cells are prepared for live-cell imaging by the confocal microscope in a 37°C incubation chamber with 5% CO2.
Mitochondrial membrane potential
Mitochondrial membrane potential
Tetramethylrhodamine ethyl ester (TMRE) reagent (Abcam) accumulates in functional and
polarized mitochondria according to Δψm.
The TMRE reagent is reconstituted in DMSO for a stock solution at 1 mM and stored at -20°C.
When tNeurons are in culture for 5 weeks in a 96-well plate, the cells are pre-treated with DMSO, 50 μM FCCP or 0.25 mM LLOME for 10 min.
Then TMRE reagent is added to fresh cell culture medium at a dilution of 1:1000 along with FCCP or LLOME. Half of the old culture medium is replaced with the TMRE-containing medium in order to incubate the cells with TMRE at a final concentration of 500 nM for 30 min at 37°C.
Cells are rinsed with pre-warmed 0.2% bovine serum albumin (BSA)/PBS twice and positioned in the CLARIOstar plate reader for fluorescence measurements.