TITLE: Microscopy-based pUb-coverage measurements of mitochondria in iNeurons
AUTHORS: Felix Kraus

[DESCRIPTION]
Microscopy-based pUb-coverage measurements of mitochondria in iNeurons

[STEPS]
SECTION: Differentiation of iNeurons
1. Day 0: Treat AAVS1-TRE3G-NGN2 cells with Accutase and plate the dissociated cells in matrigel-coated 6-well plates (2x105 cells/well) in ND1 Medium supplemented with Y27632 (10 μM).  
ND1 Medium:  
DMEM/F12 
N2 (100x)        1x  
BDNF              10 ng/ml  
NT3                 10 ng/ml  
NEAA (100X) 1x  
Laminin           0.2 μg/ml  
Doxycycline    2 μg/ml
SECTION: Differentiation of iNeurons
2. Day 1: Replace the medium with ND1 Medium.
SECTION: Differentiation of iNeurons
3. Day 2: Replace the medium with ND2 Medium.  
ND2 Medium  
Neurobasal medium  
B27 (50x)                    1x  
GlutaMax (100x)         1x  
BDNF                          10 ng/ml  
NT3                             10 ng/ml  
Doxycycline                2 μg/ml
SECTION: Differentiation of iNeurons
4. Day 4: Exchange 50% of the medium from each well.
SECTION: Differentiation of iNeurons
5. Day 6: Treat the cells with Accutase and replate the dissociated cells in matrigel-coated  6-/12-well glass bottom plates (2-4x105 cells/well for 6 wells) in ND2 Medium.
SECTION: Differentiation of iNeurons
6. Day 8 and thereafter: Exchange 50% of the medium from each well every other day.  ·       
Doxycycline can be withdrawn on Day.
SECTION: Differentiation of iNeurons
7. Induce mitophagy using Antimycin A / Oligomycin A for the desired time.
SECTION: Staining
8. Aspirate ND2 and fix cells in 1 ml pre-warmed 4% PFA for 30 min.
SECTION: Staining
9. Aspirate PFA solution and wash wells 3x with PBST (1x PBS, 0.02% Tween 20)
SECTION: Staining
10. Permeabilize the cells by adding 0.2% Triton X-100 in PBS.
SECTION: Staining
11. Remove the detergent solution by aspiration.  Wash wells 3x with PBST (1x PBS, 0.02% Tween 20). Drain well.
SECTION: Staining
12. Block cells for 10 min with 3% BSA – 1x PBS.
SECTION: Staining
13. Remove BSA solution by aspiration.  Wash wells 3x with PBST (1x PBS, 0.02% Tween 20). Drain well.
SECTION: Staining
14. Incubate with primary antibodies in 3% BSA - 1x PBS for 3h at RT with gentle shaking.
SECTION: Staining
14.1. Anti-pUb (rabbit)
SECTION: Staining
15. Wash wells 3x with PBST (1x PBS, 0.02% Tween 20). Drain well.
SECTION: Staining
16. Incubate with secondary antibodies in 3% BSA - 1x PBS for 45 min – 1h.
SECTION: Staining
16.1. Goat anti-rabbit AlexaFlour 568
SECTION: Staining
16.2. Goat anti-mouse AlexaFluor 647
SECTION: Staining
17. Wash wells 3x with PBST (1x PBS, 0.02% Tween 20). Drain well.
SECTION: Staining
18. Add Hoechst33342 or DAPI 1:2000 to wells for 5 min with gentle shaking.
SECTION: Staining
19. Wash wells 3x with PBST (1x PBS, 0.02% Tween 20). Drain well.
SECTION: Staining
20. Exchange PBST with 1x PBS and keep cells at 4°C until imaging. Image within the next few days.
SECTION: Evaluation
23. Perform image quantification was in your tool of choice. Here we will use ImageJ/FiJi and custom-written batch-macros (https://github.com/harperlaboratory/FBXO7).
SECTION: Evaluation
24. Filter mitochondrial signal (Gaussian Blur, sigma=2) and converted images into binary files and holes in the resulting mask filled.
SECTION: Evaluation
25. Threshold pUb channel into a binary file (Triangle method) and measure binary file these masks were using the "Analyze Particles..." command (pixel size exclusion: 0.5-∞, exclude edge objects).
SECTION: Evaluation
26. Save results for each image-stack (both mito and pUb) as .csv files, together with the original overlay .tiff file for QC purposes.
SECTION: Evaluation
27. Count number of nuclei for normalization.
SECTION: Evaluation
28. Plot results in your tool of choice for graphing and statistical analysis.
SECTION: Evaluation
29. Calculate the % of mitochondrial pUb coverage was and normalized to [t]=6h AO
SECTION: Fixed-cell microscopy
22. Image adequate number of cells per repeat in each condition. 
SECTION: Fixed-cell microscopy
21. Mount glass bottom plate on Yokogawa CSU-W1 spinning disk confocal on a Nikon Eclipse Ti-E motorized microscope equipped with a Nikon Apochromat 60×/1.42 N.A oil-objective lens. Image signals of 405/488/568/647 fluorophores in sequential manner with a Nikon LUN-F XL solid state laser combiner ([laser line – laser power]: 405 - 80mW, 488 - 80mW, 561 - 65mW, 640nm - 60mW]) using a Semrock Di01-T405/488/568/647 dichroic mirror. Fluorescence emissions were collected with Chroma ET455/50m [405 nm], 488 Chroma ET525/50m [488 nm], 568 Chroma ET605/52m [561 nm], 633 Chroma ET705/72m [640 nm] filters, respectively (Chroma Technologies) using NIS-Elements image acquisition software. Consistent laser intensity and exposure times must be maintained for all samples. Acquire 8 µm z-stacks for each image.
SECTION: Staining
14.2. Anti-HSP60 (mouse)