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

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 100µL /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 conditions37°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. 

Step 1: (bafilomycin treatment and fixation)  

1. To treat the cells, cell culture media is replaced with 150mL media containing 400nanomolar (nM) bafilomycin A1, and incubated at 37°C, 5%CO2 for 4h 0m 0s.  

2. After 4h cells are fixed in 2 steps to avoid detachment: 

3. Remove75µL of the culture media and replace with the same volume of 4% PFA, incubate atRoom temperature in the dark for0h 10m 0s .  

4. Remove mixture of cell culture media and PFA gently and replace with70µL of 4% PFA and incubate for0h 15m 0s . 

5. Remove PFA solution and gently wash with 1x PBS. 

Cells can be stored in PBS at 4°Cbefore 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). 

Step 2 staining with primary antibodies : 

1. Discard 1x PBS solution from wells and add permeabilization buffer 100µL per well), incubate for0h 20m 0s . 

2. Discard permeabilization buffer and add blocking buffer 100µL per well), incubate for1h 0m 0s . 

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 for0h 5m 0s (3 times) 

5. Add secondary antibodies diluted (1:500) in blocking buffer to cells 100µL per well), incubate for1h 0m 0s aRoom temperature  

6. Wash cells with 1x PBS fo0h 5m 0s (2 times). 

7. Add 1x PBS with DAPI, incubate for at least0h 7m 0s . 

8. Wash cells with 1x PBS, leave in200µL 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.