Labeling with MitoTracker™ Green FM and tetramethylrhodamine methyl ester

Cells were washed once with loading buffer containing in mM: 2 CaCl₂, 135 NaCl, 5 KCl, 1 MgCl₂, 1 HEPES, 2.6 NaHCO₃, 0.44 KH₂PO₄, 0.34 Na₂HPO₄, 10 d-glucose (Carl Roth, Karlsruhe, Germany), 0.1% vitamins, 0.2% essential amino acids and 1% penicillin/streptomycin at pH 7.4. Cells were incubated in loading buffer containing 1000, 500, 200, 81, 40.5, 13.5, 5.4, 2.7, or 1.35 nM TMRM (tetramethylrhodamine methyl ester, Invitrogen™) for 30 min. As TMRM might degrade over time in storage, TMRM concentrations were measured regularly. Therefore, after TMRM was dissolved in methanol the absorption at 550 nm was measured and the concentration was calculated using Eq. 1.

c is the molar concentration of TMRM, A the absorption of TMRM at 550 nm, ε is the specific extinction coefficient of TMRM at 550 nm in methanol, and l is the pathlength the light has to path through the TMRM solution.