F 7100 spectrofluorometer

To quantify the stimulation effects of spermine, 2 × 10⁷ HEK cells were washed in 10 mL of a wash buffer (120 mM KCl, 25 mM HEPES, 2 mM KH₂PO₄, 1 mM MgCl₂, 50 mM EGTA, pH 7.2-KOH), and resuspended in 2 mL of a recording buffer (120 mM KCl, 25 mM HEPES, 2 mM KH₂PO₄, 1 mM MgCl₂, 5 mM succinate, pH 7.2-KOH). The sample was placed in a stirred quartz cuvette in a Hitachi F-7100 spectrofluorometer (ex: 494nm, ex-slit: 2.5 nm, em: 516 nm, em-slit: 5.0 nm, sampling frequency: 1 Hz). After adding 0.25 μM of Fluo-4 (Invitrogen, F14200) to monitor [ ${\text{Ca}}^{2+}$ ]_ex, 30 mM of digitonin (Sigma, D141) was added to permeabilize the cells. When [ ${\text{Ca}}^{2+}$ ]_ex reached a steady state, spermine was added to induce further mitochondrial ${\text{Ca}}^{2+}$ absorption. At the end of the recording, 40 μM of CaCl₂ was added to obtain the saturating fluorescence ( ${\mathrm{F}}_{\max }$ ), followed by adding 500 μM of EGTA to obtain the minimum fluorescence signal ( ${\mathrm{F}}_{\text{min}}$ ). [ ${\text{Ca}}^{2+}$ _ex at a certain fluorescence signal ( $\mathrm{F}$ ) was calculated using the following equation using a Fluo-4 ${\text{Ca}}^{2+}$ K_d of 345 nM as provided by the manufacturer:
$$[C{a}^{2+}{]}_{ex}=K_d\times (F-F_{min})÷(F_{max}-F_{min})$$
To measure the rate of mitochondrial calcium uptake rate (Fig. 3D-E and 7A), Calcium Green 5N (Invitrogen, C3737) was used to monitor [ ${\text{Ca}}^{2+}$ ]_ex. Quantification was done by calculating the slope of the first 10 s of fluorescence signal decline after adding ${\text{Ca}}^{2+}$ .

2 × 10⁷ HEK cells were incubated with 40 nM of TMRM (Invitrogen, T668) in Tyrode’s solution (130 mM NaCl, 5.4 mM KCl, 1 mM MgCl₂, 1 mM CaCl₂, 20 mM HEPES, pH 7.8-NaOH) at 37 °C for 30 min. Cells were washed and pelleted down using 10 mL of a Mg²⁺-free wash buffer (120 mM KCl, 2 mM K₂HPO₄, 50 μM EGTA, 25 mM HEPES, pH 7.2-KOH) and resuspended using a 2 mL Mg²⁺-free recording buffer (120 mM KCl, 2 mM K₂HPO₄, 5 mM succinate, 25 mM HEPES, pH 7.6-KOH). The sample was loaded in a stirred quartz cuvette in a Hitachi F-7100 spectrofluorometer (excitation: 573 nm; excitation slit: 5 nm; emission: 590 nm; emission slit: 5 nm; sampling rate: 1 Hz). After the cells were permeabilized by 30 mM of digitonin, 1 mM of spermine was added to the cuvette, followed by adding 1 μg/mL of FCCP to completely collapse the IMM potential. The rate of IMM depolarization was quantified by normalizing the slope before or after adding spermine to the total TMRM signal.