Basic Procedure
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FCCP Optimization with the XF Cell Mito Stress Test
(Note: For this assay, seed cells at the optimal cell number and use the optimal oligomycin concentration
that was determined in the previous experiments.)
The XF Cell Mito Stress Test is run with six different concentrations of FCCP to determine the optimal
FCCP concentration to use in your XF assays. In a typical FCCP optimization assay, it is not necessary to
inject rotenone and antimycin A. However, for the purposes of providing richer data for discussion, we
will run the XF Cell Mito Stress Test and inject (A) oligomycin (optimal concentration), (B) FCCP (six
different concentrations) and (C) rotenone/antimycin A.
Plate Layout:
[FCCP]
0 µM 0.125 µM 0.25 µM 0.5 µM 1.0 µM 2.0 µM
Injections:
All compounds will be made at 10x the final concentrations in the wells.
Port A: oligomycin
Port A: oligomycin:
µM (optimal) final concentration in the well
Port B: FCCP –
Column 1: 0 µM final concentration in the well
Column 2: 0.125 µM final concentration in the well
Column 3: 0.25 µM final concentration in the well
Column 4: 0.50 µM final concentration in the well
Column 5: 1.0 µM final concentration in the well
Column 6: 2.0 µM final concentration in the well
Port C: rotenone/antimycin A: 0.5 µM final concentration in the well
Содержание XF24
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