Immunocytochemical analysis

Wash HeLa cells with 1x PBS

Add Trypsin to cells for 5 min and incubate at 37°C to dissociate cells from plastic well

Resuspend cells in 1 mL DMEM media

Count cells

Seed appropriate number of cells into 6-/12-/24-well glass bottom dish

Top up glass bottom dish with either 3-/1.5/1 mL DMEM and place cells back into incubator

The next day exchange DMEM with DMEM + 2µg/ml doxycycline for 18h to induce Parkin expression.

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

Aspirate DMEM 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 or overnight at 4°C with gentle shaking.

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.

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

If required, 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.