TITLE: Microscopy-based mtDNA turnover measurements in HeLa and iNeurons
AUTHORS: Felix Kraus

[DESCRIPTION]
Protocol for microscopy-based mtDNA turnover measurements in HeLa and iNeurons

[STEPS]
SECTION: Seeding of Hela cells
1. Wash HeLa cells expressing doxycycline-inducible Parkin with 1x PBS
SECTION: Seeding of Hela cells
2. Add Trypsin to cells for 5 min and incubate at 37°C to dissociate cells from plastic well
SECTION: Seeding of Hela cells
3. Resuspend cells in 1 mL DMEM media
SECTION: Seeding of Hela cells
4. Count cells
SECTION: Seeding of Hela cells
5. Seed appropriate number of cells into 24-well glass bottom dish
SECTION: Seeding of Hela cells
6. Top up glass bottom dish with either 1 mL DMEM and place cells back into incubator
SECTION: Seeding of Hela cells
7. The next day exchange DMEM with DMEM + 2µg/ml doxycycline for 18h to induce Parkin expression.
SECTION: Seeding of Hela cells
8. Induce mitophagy using Antimycin A / Oligomycin A for the desired time.
SECTION: Differentiation of iNeurons
9. 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
10. Day 1: Replace the medium with ND1 Medium.
SECTION: Differentiation of iNeurons
11. 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
12. Day 4: Exchange 50% of the medium from each well.
SECTION: Differentiation of iNeurons
13. 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
14. 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  ·       Doxycycline can be withdrawn on Day.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
15. Induce mitophagy using Antimycin A / Oligomycin A for the desired time.   
SECTION: Staining
16. Aspirate media and fix cells in 1 ml pre-warmed 4% PFA for 30 min.
SECTION: Staining
17. Aspirate PFA solution and wash wells 3x with PBST (1x PBS, 0.02% Tween 20)
SECTION: Staining
18. Permeabilize the cells by adding 0.2% Triton X-100 in PBS.
SECTION: Staining
19. Remove the detergent solution by aspiration.  Wash wells 3x with PBST (1x PBS, 0.02% Tween 20). Drain well.
SECTION: Staining
20. Block cells for 10 min with 3% BSA – 1x PBS.
SECTION: Staining
21. Remove BSA solution by aspiration.  Wash wells 3x with PBST (1x PBS, 0.02% Tween 20). Drain well.
SECTION: Staining
22. Incubate with primary antibodies in 3% BSA - 1x PBS for 3h at RT with gentle shaking.  
a.     Anti-DNA (mouse)
SECTION: Staining
23. Wash wells 3x with PBST (1x PBS, 0.02% Tween 20). Drain well.
SECTION: Staining
24. Incubate with secondary antibodies in 3% BSA - 1x PBS for 45 min – 1h.  a.     Goat anti-mouse AlexaFlour 488
SECTION: Staining
25. Wash wells 3x with PBST (1x PBS, 0.02% Tween 20). Drain well.
SECTION: Staining
26. Add Hoechst33342 or DAPI 1:2000 to wells for 5 min with gentle shaking.
SECTION: Staining
27. Wash wells 3x with PBST (1x PBS, 0.02% Tween 20). Drain well.
SECTION: Staining
28. Exchange PBST with 1x PBS and keep cells at 4°C until imaging. Image within the next few days.
SECTION: Fixed-cell microscopy
29. Mount glass bottom plate on Yokogawa CSU-W1 spinning disk confocal on a Nikon Eclipse Ti-E motorized microscope equipped with a Nikon Plan Apo 100×/1.45 N.A oil-objective lens. Image signals of 488/568/647 fluorophores in sequential manner with a Nikon LUN-F XL solid state laser combiner ([laser line – laser power]: 488 - 80mW, 561 - 65mW, 640nm - 60mW]) using a Semrock Di01-T405/488/568/647 dichroic mirror. Fluorescence emissions were collected with 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: Fixed-cell microscopy
30. Image adequate number of cells per repeat in each condition. 
SECTION: Evaluation
31. 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
32. Filter nuclear signal (Gaussian Blur, sigma=2) and converted images into binary files.
SECTION: Evaluation
33. Convert aDNA into binary files.
SECTION: Evaluation
34. Subtract the nuclear signal from the aDNA, resulting in an image containing only the mtDNA intensities.
SECTION: Evaluation
35. Measure binary file these masks were using the "Analyze Particles..." command (pixel size exclusion: 0.05-3, exclude edge objects)).
SECTION: Evaluation
36. Save results image stacks as .csv files, together with the original overlay.tiff file for QC purposes.
SECTION: Evaluation
37. Count number of nuclei for normalization.
SECTION: Evaluation
38. Plot results in your tool of choice for graphing and statistical analysis.