were coated with Laminin solution in L15+Bicarbonate: final concentration of 10 μg/mL .

Day 35 midbrain organoids were placed in the plates and media was changed every other day.

On the day of the assay supplemented with B27 + N2 was warmed at 37°C and the pH as adjusted to 7.4. And 175 ul was added to the organoid plates.

The plate containing the organoids were warmed at 37°C in a non-CO2 incubator for 30 min

Using the kit the cartridges were loaded with loaded with Glucose (10mM), Oligomicyn (1uM) and 2-DG (50mM).

The run was done in

Equipment

Value	Label
Seahorse XFe96 Analyzer	NAME
Agilent	BRAND
BLE2100298	SKU

After the run the organoids were lysed in RIPA buffer and total protein was quantified using BCA kit.

The total protein was used to normalize the ECAR and OCR.