Yeast Seahorse XF96 OCR protocol

Day 1:

Prepare the sensor cartridge with calibrant solution, 200 µl per well and incubate overnight at 30C in a CO2 free incubator. Seal with parafilm to prevent evaporation.

Make o/n cultures.

Day 2:

Start yeast cultures and grow to desired OD.

Coat the cell plate in poly-L-lysine - use 20 µl of 0.1 mg/ml per well.
Leave at room temperature for 10 minutes.
Aspirate solution and dry plate for 30 minutes.

Resuspend yeast in assay media (see below for recipe).

Add 180 µl of yeast culture to each well at an appropriate dilution. You need to titrate the cell number to give sensible and linear readings on the Seahorse, I think we used around 500000 cells per well. I do a dilution series as the first experiment to check linearity of readings.

Spin the plate for 3 min at 500 rpm to collect cells
Rest for 30 minutes at 30C
Load sensor cartridge and calibrate Seahorse probes
Measure plate at 30C as:

Mix – 2 minutes
Measure – 2 minutes
Repeat as many times as you need. I do a minimum of three measurements.

Yeast assay media:

0.167% yeast nitrogen base

0.5% ammonium sulphate

carbon source as required