TITLE: Microscopy-based evaluation of Parkin translocation and mitophagy in FBXO7-/- cell lines
AUTHORS: Felix Kraus

[DESCRIPTION]
Microscopy-based evaluation of Parkin translocation and mitophagy in FBXO7-/- cell lines

[STEPS]
SECTION: Seeding of HeLa
1. Wash HeLa cells expressing GFP-Parkin with 1x PBS
SECTION: Seeding of HeLa
2. Add Trypsin to cells for 5 min and incubate at 37°C to dissociate cells from plastic well
SECTION: Seeding of HeLa
3. Resuspend cells in 1 mL DMEM media
SECTION: Seeding of HeLa
4. Count cells
SECTION: Seeding of HeLa
5. Seed appropriate number of cells into 24-well glass bottom dish
SECTION: Seeding of HeLa
6. Top up glass bottom dish with either 1 mL DMEM and place cells back into incubator
SECTION: Seeding of HeLa
7. Induce mitophagy using Antimycin A / Oligomycin A for the desired time.
SECTION: Staining
8. Aspirate DMEM 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.  a.     Anti-GFP (chicken)  b.     Anti-HSP60 (mouse)
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.  a.     Goat anti-chicken AlexaFlour 488  b.     Goat anti-mouse AlexaFluor 647  c.     DNA-SPY555
SECTION: Staining
17. Wash wells 3x with PBST (1x PBS, 0.02% Tween 20). Drain well.
SECTION: Staining
18. Wash wells 3x with PBST (1x PBS, 0.02% Tween 20). Drain well.
SECTION: Staining
19. Exchange PBST with 1x PBS and keep cells at 4°C until imaging. Image within the next few days.
SECTION: Fixed-cell microscopy
20. 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 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
21. Image adequate number of cells per repeat in each condition.
SECTION: Evaluation
22. Perform image quantification was in your tool of choice. Here we will use CellProfiler and segmentation pipelines can be found online (https://github.com/harperlaboratory/FBXO7).
SECTION: Evaluation
23. Export results and plot in your tool of choice for graphing and statistical analysis.