TITLE: Evaluation of pUb kinetics using 3D-SIM
AUTHORS: Felix Kraus

[DESCRIPTION]
Protocol for the evaluation of pUb kinetics using 3D-SIM

[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 onto 18x18mm Marienfeld Precision cover glasses thickness No. 1.5H (tol. ± 5 μm).
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  18x18mm Marienfeld Precision cover glasses thickness No. 1.5H (tol. ± 5 μm).
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Doxycycline can be withdrawn on Day.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 DMEM and fix cells in warm paraformaldehyde 3% Glutaraldehyde 0.35% in 0.1M Sodium Cacodylate, pH 7.4Aspirate PFA solution and wash wells 3x with PBST (1x PBS, 0.02% Tween 20)
SECTION: Staining
17. Permeabilize the cells by adding 0.2% Triton X-100 in PBS.
SECTION: Staining
18. Remove the detergent solution by aspiration.  Wash wells 3x with PBST (1x PBS, 0.02% Tween 20). Drain well.
SECTION: Staining
19. Block cells for 10 min with 3% BSA – 1x PBS.
SECTION: Staining
20. Remove BSA solution by aspiration.  Wash wells 3x with PBST (1x PBS, 0.02% Tween 20). Drain well.
SECTION: Staining
21. Incubate with primary antibodies in 3% BSA - 1x PBS over night at 4°C with gentle shaking.  
a.     Anti-HSP60 (mouse)  
b.     Anti-pUb (rabbit)
SECTION: Staining
22. Wash wells 3x with PBST (1x PBS, 0.02% Tween 20). Drain well.
SECTION: Staining
23. Incubate with secondary antibodies in 3% BSA - 1x PBS for 45 min – 1h.  
a.     Goat anti-mouse AlexaFlour 488  
b.     Goat anti-rabbit AlexaFluor 568
SECTION: Staining
24. Wash wells 3x with PBST (1x PBS, 0.02% Tween 20). Drain well.
SECTION: Staining
25. Add DAPI 1:2000 to wells for 5 min with gentle shaking.
SECTION: Staining
26. Wash wells 3x with PBST (1x PBS, 0.02% Tween 20). Drain well.
SECTION: Staining
27. Wash coverslips with 1x PBS and mount in Vectashield (Vector Laboratories, H-1000-10) on glass slides.Exchange PBST with 1x PBS and keep cells at 4°C until imaging. Image within the next few days. 
SECTION: Fixed-cell 3D-SIM microscopy
28. Image cells on DeltaVision OMX v4 using an Olympus 60x / 1.42 Plan Apo oil objective (Olympus, Japan). Record 405, 488 and 568 channels using a front-illuminated sCMOS (PCO Photonics, USA) in 512x512px image size mode, 1x binning, 125 nm z-stepping and with 15 raw images taken per z-plane (5 phase-shifts, 3 angles).
SECTION: Fixed-cell 3D-SIM microscopy
29. Reconstruct raw images using CUDA-accelerated 3D-SIM reconstruction code (https://github.com/scopetools/cudasirecon) based on Gustafsson et al. (2008[FK1] ). The Optimal optical transfer function (OTF) was determined via an in-house build software, developed by Talley Lambert from the NIC / CBMF (GitHub: https://github.com/tlambert03/otfsearch, all channels were registered to the 528nm output channel, Wiener filter: 0.002, background: 90).   
SECTION: Evaluation
30. Import composite .dv stacks into Imaris (v9.7) and convert into native .ims files.
SECTION: Evaluation
31. Import into Imaris Arena and perform global background subtraction.
SECTION: Evaluation
32. Segment pUb and mitochondrial objects from seeds (XY starting diameter: 0.08µm == pixel size of images), segmented based on automatic thresholding with local background subtraction and splitting of touching objects (0.4µm).
SECTION: Evaluation
33. Pipe segmented objects into Imaris Vantage module for further analysis.
SECTION: Evaluation
34. In Vantage, compute nearest neighbour distances of pUb to pUb and between pUb and mitochondria, as well as volume of segmented objects.
SECTION: Evaluation
35. Test pipeline on WT control cells and then applied for batch processing on all other genotype to allow for unbiased segmentation and analysis.
SECTION: Evaluation
36. Plot results in your tool of choice for graphing and statistical analysis.