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

[DESCRIPTION]
Protocol for microscopy-based pUb-coverage measurements of mitochondria in iNeurons

[STEPS]
SECTION: Differentiation of iNeurons
22. 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
22. Day 1: Replace the medium with ND1 Medium.
SECTION: Differentiation of iNeurons
22. 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
22. Day 4: Exchange 50% of the medium from each well.
SECTION: Differentiation of iNeurons
22. Day 6: Treat the cells with Accutase and replate the dissociated cells in matrigel-coated
SECTION: Differentiation of iNeurons
22. 6-/12-well glass bottom plates (2-4x105 cells/well for 6 wells) in ND2 Medium.
SECTION: Differentiation of iNeurons
22. 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  ·       Doxycycline can be withdrawn on Day.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
22. Mount glass bottom plate on Yokogawa CSU-W1 spinning disk confocal on a Nikon Eclipse Ti-E motorized microscope. Ensure that the system is equipped with a Tokai Hit stage top incubator and imaging was performed at 37°C, 5% CO2 and 95% humidity.
SECTION: Differentiation of iNeurons
22. Take image stacks using a Nikon Plan Apo 60×/1.40 N.A immersion oil objective lens.
SECTION: Differentiation of iNeurons
22. For ratiometic imaging, mtKeimaXL were excited in sequential manner with a Nikon LUN-F XL solid state laser combiner ([laser line – laser power]: 445 - 80mW, 561 – 65 mW]) using a Semrock Di01-T445/515/561 dichroic mirror. Fluorescence emissions were collected through a Chroma ET605/52m [for 445 nm] and a 568 Chroma ET605/52m [for 561 nm], filters, respectively (Chroma Technologies). Consistent laser intensity and exposure times must be maintained for all samples.
SECTION: Live-cell microscopy
22. Mount glass bottom plate on Yokogawa CSU-W1 spinning disk confocal on a Nikon Eclipse Ti-E motorized microscope. Ensure that the system is equipped with a Tokai Hit stage top incubator and imaging was performed at 37°C, 5% CO2 and 95% humidity.
SECTION: Live-cell microscopy
22. Take image stacks using a Nikon Plan Apo 60×/1.40 N.A immersion oil objective lens.
SECTION: Live-cell microscopy
22. For ratiometic imaging, mtKeimaXL were excited in sequential manner with a Nikon LUN-F XL solid state laser combiner ([laser line – laser power]: 445 - 80mW, 561 – 65 mW]) using a Semrock Di01-T445/515/561 dichroic mirror. Fluorescence emissions were collected through a Chroma ET605/52m [for 445 nm] and a 568 Chroma ET605/52m [for 561 nm], filters, respectively (Chroma Technologies). Consistent laser intensity and exposure times must be maintained for all samples.
SECTION: Evaluation
22. 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
22. Divide raw confocal images of mitochondrial targeted mt-mKeimaXL [ex:561/ex:445],  resulting in a ratiometic image of only acidic Keima-puncta.
SECTION: Evaluation
22. Subject resulting signals to background subtraction (rolling kernel size 25, sliding paraboloid) and convert into binary objects.
SECTION: Evaluation
22. Use the "Analyze Particles..." command (pixel size exclusion: 0.5-∞, exclude edge objects) to measure foci-abundance and other morphological parameters.
SECTION: Evaluation
22. Save results for each image-stack as .csv files, together with the original ratiometic .tiff file for QC purposes.
SECTION: Evaluation
22. Count number of nuclei for normalization.
SECTION: Evaluation
22. Plot results in your tool of choice for graphing and statistical analysis.