Seahorse XF assay

Question 1

Which 4 reagents are used in the Mito Stress assay in sequential order

Answer 1

Oligomycin
FCCP
Rotenone and Antimycin A

Question 2

Function of oligomycin

Answer 2

Inhibits ATP Synthase
Determines: ATP-linked Respiration and Proton Leak
Brings down OCR so that the DIFFERENCE between the basal OCR and new OCR is a measure of ATP-linked Respiration, and the OCR below the curve/level is due to proton leak

Question 3

Function of FCCP

Answer 3

FCCP = uncoupling agent - targets inner mitochondrial membrane
Collapses proton gradient, maximises OCR
FCCP-stimulated OCR calculates spare respiratory capacity

Question 4

What is the spare respiratory capacity

Answer 4

Difference in OCR between max respiration and basal respiration
Measure of ability of cell to respond to increased energy demand or under stress

Question 5

Functions of Rotenone and Antimycin A

Answer 5

Rotenone - complex I inhibitor
Antimycin A - Complex III inhibitor
Combination shuts down mitochondrial respiration, enables calculation of nonmitochondrial respiration driven by processes outside of mitrochondria