Microscopy-based mitochondrial morphology measurements in iNeurons

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

Day 1: Replace the medium with ND1 Medium.

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

Day 4: Exchange 50% of the medium from each well.

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.

Day 8 and thereafter: Exchange 50% of the medium from each well every other day. ·

Doxycycline can be withdrawn on Day.

Induce mitophagy using Antimycin A / Oligomycin A for the desired time.

Aspirate medium and fix cells in 1 ml pre-warmed 4% PFA for 30 min.

Aspirate PFA solution and wash wells 3x with PBST (1x PBS, 0.02% Tween 20)

Permeabilize the cells by adding 0.2% Triton X-100 in PBS.

Remove the detergent solution by aspiration. Wash wells 3x with PBST (1x PBS, 0.02% Tween 20). Drain well.

Block cells for 10 min with 3% BSA – 1x PBS.

Remove BSA solution by aspiration. Wash wells 3x with PBST (1x PBS, 0.02% Tween 20). Drain well.

Incubate with primary antibodies in 3% BSA - 1x PBS for 3h at RT with gentle shaking.

a. Anti-HSP60 (mouse)

Wash wells 3x with PBST (1x PBS, 0.02% Tween 20). Drain well.

Incubate with secondary antibodies in 3% BSA - 1x PBS for 45 min – 1h.

b. Goat anti-mouse AlexaFlour 488

Wash wells 3x with PBST (1x PBS, 0.02% Tween 20). Drain well.

Add Hoechst33342 or DAPI 1:2000 to wells for 5 min with gentle shaking.

Wash wells 3x with PBST (1x PBS, 0.02% Tween 20). Drain well.

Exchange PBST with 1x PBS and keep cells at 4°C until imaging. Image within the next few days.

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.

Image adequate number of cells per repeat in each condition.

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).

Count number of nuclei for normalization.

Plot results in your tool of choice for graphing and statistical analysis.