Treatment and staining of iPSC-derived neurons for lysosomal phenotype analysis
Jessica Chedid, Adahir Labrador-Garrido, Nicolas Dzamko
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.
Attachments
Steps
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 (100µL/well).
Image the cells for 0h 15m 0s, 0h 45m 0s and 1h 30m 0s after adding the probes using the Opera Phenix high-content screening system.
Fixed cells experimental outline-Bafilomycin treatment and fixation
To treat the cells, replace the cell culture media with 150µL media containing 400nanomolar (nM)bafilomycin A1, and incubate at 37°C, 5% CO2 for 4h 0m 0s.
Fix the cells after 4h 0m 0s in 2 steps to avoid detachment.
Remove 75µL of the culture media and replace with the same volume of 4% PFA, incubate at Room temperature in the dark for 0h 10m 0s.
Remove mixture of cell culture media and PFA gently and replace with 70µL of 4% PFA and incubate for 0h 15m 0s.
Remove PFA solution and gently wash with 1x PBS.
Store the cells in PBS at 4°C before commencing staining.
Fixed cells experimental outline-Staining with primary antibodies
Discard 1x PBS solution from wells and add permeabilization buffer (100µL per well), incubate for
0h 20m 0s.
Discard permeabilization buffer and add blocking buffer (100µL per well), incubate for 1h 0m 0s.
Prepare antibody combinations to desired final concentrations in blocking buffer, discard blocking buffer from plates and replace with primary antibody dilutions, incubate 1h 0m 0s at 4°C.
Wash cells with 1x PBS for 5 min (3 times).
Wash cells with 1x PBS for 0h 5m 0s (1/3).
Wash cells with 1x PBS for 0h 5m 0s (2/3).
Wash cells with 1x PBS for 0h 5m 0s (3/3).
Add secondary antibodies diluted (1:500) in blocking buffer to cells (100µLper well), incubate for 1h 0m 0s at Room temperature.
Wash cells with 1x PBS for 5 min (2 times).
Wash cells with 1x PBS for 0h 5m 0s (1/2).
Wash cells with 1x PBS for 0h 5m 0s (2/2).
Add 1x PBS with DAPI, incubate for at least 0h 7m 0s.
Wash cells with 1x PBS, leave in 200µL of 1x PBS per well to avoid drying out.
Plates are now ready to be imaged.
