TITLE: Treatment and staining of iPSC-derived neurons for lysosomal phenotype analysis
AUTHORS: Jessica Chedid, Adahir Labrador-Garrido, Nicolas Dzamko

[DESCRIPTION]
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]

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.


[STEPS]
SECTION: Live cells 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.
SECTION: Live cells experimental outline
2. Alternatively prepare: Mitotracker 1:10.000, Lysosomal staining 1:500, Cytopainter 1:500 and Hoechst 1:100 in complete cell culture media.
SECTION: Live cells experimental outline
3. Gently replace cell culture media on the cells with the prepared solution (100 µL/well).

SECTION: Live cells experimental outline
4. Image the cells for   15 min, 45 min and 90 min after adding the probes using the Opera Phenix high-content screening system. 
 

SECTION: Fixed cells experimental outline-Bafilomycin treatment and fixation
5. 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 240 min.
SECTION: Fixed cells experimental outline-Staining with primary antibodies
11. Discard 1x PBS solution from wells and add permeabilization buffer (100 µL per well), incubate for 
 20 min.
SECTION: Fixed cells experimental outline-Bafilomycin treatment and fixation
6. Fix the cells after 240 min  in 2 steps to avoid detachment.
SECTION: Fixed cells experimental outline-Bafilomycin treatment and fixation
9. Remove PFA solution and gently wash with 1x PBS.
SECTION: Fixed cells experimental outline-Bafilomycin treatment and fixation
8. Remove mixture of cell culture media and PFA gently and replace with 70 µL of 4% PFA and incubate for 15 min.
SECTION: Fixed cells experimental outline-Bafilomycin treatment and fixation
7. Remove 75 µL of the culture media and replace with the same volume of 4% PFA, incubate at Room temperature in the dark for 10 min. 
SECTION: Fixed cells experimental outline-Bafilomycin treatment and fixation
10. Store the cells in PBS at 4 °C before commencing staining.
 

SECTION: Fixed cells experimental outline-Staining with primary antibodies
12. Discard permeabilization buffer and add blocking buffer (100 µL per well), incubate for 60 min.
SECTION: Fixed cells experimental outline-Staining with primary antibodies
15. Add secondary antibodies diluted (1:500) in blocking buffer to cells (100 µLper well), incubate for 60 min at Room temperature.
SECTION: Fixed cells experimental outline-Staining with primary antibodies
14. Wash cells with 1x PBS for 5 min (3 times).
SECTION: Fixed cells experimental outline-Staining with primary antibodies
13. Prepare antibody combinations to desired final concentrations in blocking buffer, discard blocking buffer from plates and replace with primary antibody dilutions, incubate 60 min at  4 °C.
SECTION: Fixed cells experimental outline-Staining with primary antibodies
14.1. Wash cells with 1x PBS for 5 min (1/3).
SECTION: Fixed cells experimental outline-Staining with primary antibodies
14.3. Wash cells with 1x PBS for 5 min (3/3).
SECTION: Fixed cells experimental outline-Staining with primary antibodies
14.2. Wash cells with 1x PBS for 5 min (2/3).
SECTION: Fixed cells experimental outline-Staining with primary antibodies
16. Wash cells with 1x PBS for 5 min (2 times).
SECTION: Fixed cells experimental outline-Staining with primary antibodies
16.1. Wash cells with 1x PBS for 5 min (1/2).

SECTION: Fixed cells experimental outline-Staining with primary antibodies
16.2. Wash cells with 1x PBS for 5 min (2/2).
SECTION: Fixed cells experimental outline-Staining with primary antibodies
17. Add 1x PBS with DAPI, incubate for at least 7 min.
SECTION: Fixed cells experimental outline-Staining with primary antibodies
18. Wash cells with 1x PBS, leave in 200 µL of 1x PBS per well to avoid drying out.
SECTION: Fixed cells experimental outline-Staining with primary antibodies
19. Plates are now ready to be imaged.