Sep 19, 2022

Treatment and staining of iPSC-derived neurons for lysosomal phenotype analysis

DOI
https://dx.doi.org/10.17504/protocols.io.261ge3pmdl47/v1
  • Jessica Chedid1,
  • Adahir Labrador-Garrido1,
  • Nicolas Dzamko1
  • 1The University of Sydney
Benjamin Trist
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DOI: https://dx.doi.org/10.17504/protocols.io.261ge3pmdl47/v1
Protocol Citation: Jessica Chedid, Adahir Labrador-Garrido, Nicolas Dzamko 2022. Treatment and staining of iPSC-derived neurons for lysosomal phenotype analysis. protocols.io https://dx.doi.org/10.17504/protocols.io.261ge3pmdl47/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: September 06, 2022
Last Modified: May 31, 2024
Protocol  Integer ID: 69606
Keywords: Immunocytochemistry, iPSC, neurons, Live imaging, Autophagy, Lysosome, ASAPCRN, neurons for lysosomal phenotype analysis, lysosomal pathway inhibitor bafilomycin a1, quantification of autophagy measure, lysosomal phenotype analysis, dynamic readout of the autophagy state, staining of the autophagic markers p62, lysosomal activity, autophagy measure, autophagic markers p62, autophagy flux, autophagy flux in the presence, lysosomal, autophagy state, lysosome, autophagosomal phenotype, treatment of neuronal culture, absence of bafilomycin a1 treatment, bafilomycin a1 treatment, cell lines for the measurement, staining of ipsc, cell line, neuronal culture, guideline for stain
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.
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Guidelines

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:
1. Probing and imaging live cells. 2. Treating, staining, and imaging fixed cells.
Materials

Consumables:

96 well-plates from Perkin Elmer: CELLCARRIER-96 ULTRA Black with clear bottom TC treated sterile Perkin ElmerCatalog #6055300


Reagents

For live cells:

  • Cell culture media* (refer to ‘material input’ section for details)
  • DQ™ Red BSA  - Special PackagingThermo FisherCatalog #D12051
  • Lysosomal Staining Reagent- Orange-Cytopainter AbcamCatalog #ab176827
  • MitoTracker™ Deep Red FM - Special PackagingThermo FisherCatalog #M22426
  • Cell Proliferation Staining Reagent - Green Fluorescence - Cytopainter AbcamCatalog #176735
  • PureBlu™ Hoechst 33342 Nuclear Staining DyeBio-Rad LaboratoriesCatalog #1351304


For fixed cells:

  • Triton™ X-100Merck MilliporeSigma (Sigma-Aldrich)Catalog #T8787-100ML
  • Bovine serum albumin (Bovostar BSAS-AU 500g)
  • ParaformaldehydeMerck MilliporeSigma (Sigma-Aldrich)Catalog #416780010
  • Bafilomycin A1 from Streptomyces griseusMerck MilliporeSigma (Sigma-Aldrich)Catalog #B1793-10UG
  • DAPI nuclear stain


Antibodies:

ABCD
AntibodiesSpeciesSourceCat n#
MAP2*ChickenThermo FisherPA1-10005
TH*SheepThermo FisherPA1-4679
TH*RabbitThermo FisherOPA1-04050
LC3RabbitAbcamab192890
P62MouseAbcamab56416

* These antibodies are included to identify desired cell populations and can be substituted as required for different differentiation protocols.

MAP2 Polyclonal AntibodyThermo Fisher ScientificCatalog #PA1-10005
Tyrosine Hydroxylase Polyclonal AntibodyThermo Fisher ScientificCatalog #PA1-4679
Tyrosine Hydroxylase Polyclonal AntibodyThermo Fisher ScientificCatalog #OPA1-04050
Recombinant Anti-LC3B antibody [EPR18709] - Autophagosome Marker (ab192890)AbcamCatalog #ab192890
Anti-SQSTM1 / p62 antibody [2C11] - BSA and Azide freeAbcamCatalog #ab56416


Solutions:

AB
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


Material input (animal, cell, tissue, fraction details)

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 cells experimental outline
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.
Gently replace cell culture media on the cells with the prepared solution (100 µL /well).

Image the cells for 00:15:00 , 00:45:00 and 01:30:00 after adding the probes using the Opera Phenix high-content screening system.
Note
  • 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.


2h 30m
Fixed cells experimental outline-Bafilomycin treatment and fixation
12h 2m
To treat the cells, replace the cell culture media with 150 µL media containing 400 nanomolar (nM) bafilomycin A1, and incubate at 37 °C , 5% CO2 for 04:00:00 .
4h
Fix the cells after 04:00:00 in 2 steps to avoid detachment.
4h
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 .
10m
Remove mixture of cell culture media and PFA gently and replace with 70 µL of 4% PFA and incubate for 00:15:00 .
15m
Remove PFA solution and gently wash with 1x PBS.
Store the cells in PBS at 4 °C before commencing staining.
Note
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.

Fixed cells experimental outline-Staining with primary antibodies
12h 2m
Discard 1x PBS solution from wells and add permeabilization buffer (100 µL per well), incubate for
00:20:00 .
20m
Discard permeabilization buffer and add blocking buffer (100 µL per well), incubate for 01:00:00 .
1h
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 .
1h
Wash cells with 1x PBS for 5 min (3 times).
Wash cells with 1x PBS for 00:05:00 (1/3).
5m
Wash cells with 1x PBS for 00:05:00 (2/3).
5m
Wash cells with 1x PBS for 00:05:00 (3/3).
5m
Add secondary antibodies diluted (1:500) in blocking buffer to cells (100 µL per well), incubate for 01:00:00 at Room temperature .
1h
Wash cells with 1x PBS for 5 min (2 times).
Wash cells with 1x PBS for 00:05:00 (1/2).

5m
Wash cells with 1x PBS for 00:05:00 (2/2).
5m
Add 1x PBS with DAPI, incubate for at least 00:07:00 .
7m
Wash cells with 1x PBS, leave in 200 µL of 1x PBS per well to avoid drying out.
Plates are now ready to be imaged.
Note
  • 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.