The Seahorse analyser is a device that provides a metabolic phenotype to samples of living cells
Specifically it measures Oxygen Consumption Rate (OCR) as a proxy for The Kreb’s Cycle and Extracellular Acidification Rate (ECAR) as a proxy for Glycolysis
This diagram is a close up of one well in a 96 well plate. The pink area represents the small volume of media (~3µl) trapped between the 96 well plate and the cartridge. The cells this volume reduce the O2 in this media by Kreb’s/ OxPhos. and provide protons H+ by Glycolysis. These species are detected by two beads of fluorochromes that are illuminated by two different LED’s through fibres. A change in Oxygen concentration or acidity changes the fluorescence. Each well has 4 programmable injection ports (A,B,C,D) which which can be manually loaded (8 channel pipettor) with 25µl of reagent
The Seahorse is charged at £35 p/h under normoxic conditions and £150 per day for hypoxic use
|The Seahorse is located in the Respiratory Medicine lab on floor 5, (Room 05.868) all Seahorse users must undergo Cl2 room training with Ian Horan.
A non-CO2 incubator is available in this lab as the sensor cartridges must be pre-incubated (hydrated) overnight. To do this, add 200µl “Calibrant” solution to each well of the utility plate (blank version of 96 well plate), slot in the sensor cartridge and leave overnight
Note that both the Seahorse and its computer must be on to activate the 37° heater on the machine
Each well should be loaded with 80µl of cell suspension. The background correction wells (A1, A12, H1, H12) should have media only
On the day, the cells must be incubated in the CO2 free media for at least an hour
The sensor cartridges must be placed in the machine barcode first
Ports are loaded via an loading guide that prevents contamination. All wells must have some control or reagent solution in each port used.