Seahorse injection C solution

Reagent	Final solution concentration	Volume
RPMI reconstituted medium	N/A	2.5 mL
Rotenone 2.5 mM	5 μM	5 μL
Antimycin A 10 mM	5 μM	1.25 μL
Hoechst 20 mM	80 μM	10 μL
Total	N/A	2.5 mL

Seahorse injection C solution is freshly prepared before usage. Solve 1 mg Rotenone with 2.55 mL DMSO to get 100 mM Rotenone solution, which is further diluted into 2.5 mM with DMSO. Rotenone solution (2.5 mM) is aliquoted into 10 μL/tube and stored at −80°C for up to 12 months. 10 mM Antimycin is prepared by solving 25 mg Antimycin powder with 4.7 mL DMSO. Antimycin solution (10 mM) is aliquoted into 5 μL/tube and stored at −80°C for up to 12 months. 20 mM Hoechst solution can be stored at 2°C–8°C for up to 6 months.