Feb 27, 2024
Treatment and staining of iPSC-derived neurons for lysosomal phenotype analysis
- Jessica Chedid1,
- Adahir Labrador-Garrido1,
- Nicolas Dzamko1
- 1University of Sydney
Protocol Citation: Jessica Chedid, Adahir Labrador-Garrido, Nicolas Dzamko 2024. Treatment and staining of iPSC-derived neurons for lysosomal phenotype analysis. protocols.io https://dx.doi.org/10.17504/protocols.io.e6nvwdwedlmk/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 27, 2024
Last Modified: February 27, 2024
Protocol Integer ID: 95838
Funders Acknowledgement:
Michael J Fox Foundation
Grant ID: ASAP-000497
Abstract
This protocol describes the preparation and treatment of neuronal cultures to be imaged for its analysis using the Opera Phenix high-content screening system. This includes the preparation of the cultures and its treatment to stain Lysosomes using a lysosome staining reagent, the treatment with DQ-red BSA to analyse lysosomal activity and the fixation and staining of the autophagic markers P62 and LC3 in the presence and absence of the autophagy-lysosomal pathway inhibitor Bafilomycin A1. Quantification of autophagy measures or autophagy flux in the presence and absence of bafilomycin A1 treatment offers a dynamic readout of the autophagy state that cannot be captured otherwise in immunostaining and western blot experiments. The aim of this protocol is to provide a guideline for stain and image any cell line for its analysis using a high content imaging system, allowing the process of large number of conditions/cell lines for the measurement of lysosomal and autophagosomal phenotypes.
Guidelines
Please refer to the Harmony software manual (https://www.perkinelmer.com/uk/product/harmony-4-9-office-license-hh17000010) for assistance in setting imaging parameters.
Materials
Consumables
96 well-plates from Perkin Elmer: CELLCARRIER-96 ULTRA Black with clear bottom, TC treated, sterile with lid (catalogue no. 6055300)
Reagents
For live cells:
Cell culture media* (refer to ‘material input’ section for details)
DQ-red BSA (ThermoFisher D12051)
Lysosomal Staining Reagent- Orange-Cytopainter (abcam ab176827)
Mitotracker deep red (ThermoFisher M22426)
Cell proliferation staining reagent – Green fluorescence – Cytopainter (abcam ab176735)
PUREBLUTM Hoesht 33342 (Bio-Rad #1351304)
For fixed cells:
Triton X100 (Sigma #T8787-100ml)
Bovine serum albumin (Bovostar BSAS-AU 500g)
Paraformaldehyde (Sigma #ACR416780010)
Bafilomycin A1 (B1793-10UG)
DAPI nuclear stain
Antibodies:
| Antibodies | Species | Source | Cat n# | |
| MAP2* | Chicken | Thermo Fisher | PA1-10005 | |
| TH* | Sheep | Thermo Fisher | PA1-4679 | |
| TH* | Rabbit | Thermo Fisher | OPA1-04050 | |
| LC3 | Rabbit | Abcam | ab192890 | |
| P62 | Mouse | Abcam | ab56416 |
Antibodies used in this protocol
* These antibodies are included to identify desired cell populations and can be substituted as required for different differentiation protocols.
Solutions
Fixing solution: 4% PFA in 1xPBS
Blocking buffer: 3% BSA + 0.1% Triton X-100 in 1 x PBS
Permeabilization buffer: 0.3% Triton X-100 in 1 x PBS
Antibodies dilution solution: antibodies are prepared in blocking buffer
Washing solution: 1x PBS
Before start
Experimental Outline
Cells are seeded at 30-50k cells/well and maintained in cell culture media until the desired experimental endpoint. Initial plating densities should be optimized for each cell type or cell line to provide optimal survival rates, morphology, and differentiation at final timepoint.
When cells are ready to be stained, the protocol diverges into 2 separate series of steps:
- Probing and imaging live cells
- Treating, staining, and imaging fixed cells.
This protocol can be applied to different cell types for the assessment of lysosomal functions. Here we apply it to induced pluripotent stem cells differentiated into Ventral Medial Dopaminergic neurons (protocol available at 10.17504/protocols.io.bu7ynzpw) or cortical neurons (protocol available at 10.17504/protocols.io.bu6znzf6). Cortical neurons were cultured until DIV50 and ventral medial dopaminergic neurons cultured until DIV40.
Live Cell experimental outline
Live Cell experimental outline
1. Prepare: DQ-red-BSA 1:100, Cytopainter green cell proliferation reagent 1:500 and Hoechst 1:100 in complete cell culture media.
Alternatively prepare: Mitotracker 1:10.000, Lysosomal staining 1:500, Cytopainter 1:500 and Hoechst 1:100 in complete cell culture media.
2. Gently replace cell culture media on the cells with the prepared solution (100 µL /well)
3. Cells are imaged 15, 45 and 90 minutes after adding the probes using the Opera Phenix high-content screening system. Hoechst, Alexa488 and Alexa561 Laser/filter pairs are used for DQ-red BSA treatment imaging. Hoechst, Alexa488, Alexa561 and Alexa647 laser/filter pairs are used to image Mitotracker/Lysosomal probes.
Suggested Imaging conditions:
40x water objective, 3 z-steps, at least 25 fields of view, imaging done in cell culture conditions (37 °C , 5% CO2).
Note: 40x objective is needed to obtain enough detail for accurate Lysosome-Mitophagy analysis. Z-step and fields of view are selected to obtain enough images without compromising the time it takes to finish a round of imaging.
Fixed cells experimental outline
Fixed cells experimental outline
7h 2m
Step 1: (bafilomycin treatment and fixation)
1. To treat the cells, cell culture media is replaced with 150 mL media containing 400 nanomolar (nM) bafilomycin A1, and incubated at 37 °C , 5%CO2 for 04:00:00 .
2. After 4h cells are fixed in 2 steps to avoid detachment:
3. Remove 75 µL of the culture media and replace with the same volume of 4% PFA, incubate at Room temperature in the dark for 00:10:00 .
4. Remove mixture of cell culture media and PFA gently and replace with 70 µL of 4% PFA and incubate for 00:15:00 .
5. Remove PFA solution and gently wash with 1x PBS.
Cells can be stored in PBS at 4 °C before commencing staining. (At this point plates can be used for later steps of permeabilization, blocking and staining or can be stored at 4 °C in the dark for several days).
4h 25m
Step 2 staining with primary antibodies:
1. Discard 1x PBS solution from wells and add permeabilization buffer (100 µL per well), incubate for 00:20:00 .
2. Discard permeabilization buffer and add blocking buffer (100 µL per well), incubate for 01:00:00 .
3. Prepare antibody combinations to desired final concentrations in blocking buffer, discard blocking buffer from plates and replace with primary antibody dilutions, incubate overnight at 4 °C .
4. Wash cells with 1x PBS for 00:05:00 (3 times)
5. Add secondary antibodies diluted (1:500) in blocking buffer to cells (100 µL per well), incubate for 01:00:00 atRoom temperature
6. Wash cells with 1x PBS for00:05:00 (2 times).
7. Add 1x PBS with DAPI, incubate for at least 00:07:00 .
8. Wash cells with 1x PBS, leave in 200 µL of 1x PBS per well to avoid drying out.
9. Plates are now ready to be imaged.
Suggested Imaging conditions:
40x water objective, 10 z-steps (0.5mm step size as recommended by the manufacturer), at least 46 fields of view per well (covering 16% of the well’s area).
Note: Imaging conditions are selected taking into consideration the detail needed (analysis of organelles need higher magnification), and the minimum number of cells needed to obtain a robust result (if the culture has very little number of cells, more fields of view could be needed). Please refer to the Harmony software manual (https://www.perkinelmer.com/uk/product/harmony-4-9-office-license-hh17000010) for assistance in setting imaging parameters.
2h 37m