TITLE: Flow cytometry-based measurement of mitophagic flux
AUTHORS: Felix Kraus

[DESCRIPTION]
Protocol for flow cytometry-based measurement of mitophagic flux

[STEPS]
SECTION: Generation of stable mtKeima cell lines
1. Generate stable cell lines expressing mitochondrial targeted mKeima. See dx.doi.org/10.17504/protocols.io.br87m9zn and dx.doi.org/10.17504/protocols.io.6qpvr4xn3gmk/v1 
SECTION: Seeding of HeLa cells
2. Wash HeLa cells expressing doxycycline-inducible Parkin with 1x PBS
SECTION: Seeding of HeLa cells
3. Add Trypsin to cells for 5 min and incubate at 37°C to dissociate cells from plastic well
SECTION: Seeding of HeLa cells
4. Resuspend cells in 1 mL DMEM media
SECTION: Seeding of HeLa cells
5. Count cells
SECTION: Seeding of HeLa cells
6. Seed appropriate number of cells into 24-well glass bottom dish
SECTION: Seeding of HeLa cells
7. Top up glass bottom dish with either 1 mL DMEM and place cells back into incubator
SECTION: Seeding of HeLa cells
8. The next day exchange DMEM with DMEM + 2µg/ml doxycycline for 18h to induce Parkin expression.
SECTION: Seeding of HeLa cells
9. Induce mitophagy using Antimycin A / Oligomycin A for the desired time. Also have BafA (25nM) sample for normalization. 
==> proceed to step 17 for flow cytometry-based measurements. 
SECTION: Differentiation of iNeurons
10. 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
11. Day 1: Replace the medium with ND1 Medium.
SECTION: Differentiation of iNeurons
12. 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
13. Day 4: Exchange 50% of the medium from each well.
SECTION: Differentiation of iNeurons
14. 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
15. 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
16. Induce mitophagy using Antimycin A / Oligomycin A for the desired time. Also have BafA (25nM) sample for normalization. 
SECTION: Keima mito flux analysis
17. Detach cells from vessel of choice and resuspend carefully so they do not clump. If required, filtered through a cell strainer cap tube. Place in vessel of choice for cytometry, for example tubes or 96 well plates. 

SECTION: Keima mito flux analysis
18. Use dual-excitation (440nm for ph7 and 561nm for pH3) and collect in 620 nm range. Analyze at least 10.000 single, healthy cells. Calculate of acidic:neutral mt-Keima ratio on a per-cell basis in FlowJo Software.
If have BafA sample, use as normalization for sample set in genotype, otherwise use fed control.