Measuring Cytosolic and Mitochondrial Ca2+ Levels

Cytosolic and mitochondrial Ca²⁺ levels were measured following IP3R-mediated release from ER stores in HEK293 cells^{20 (link)}. To do so, cells transfected with M3R and either empty vector control plasmid or TDP-43 plasmids were loaded with either 2 μM Fluo4-AM or Rhod2-AM dye (Invitrogen) in external solution (145 mM NaCl, 2 mM KCl, 5 mM NaHCO₃, 1 mM MgCl₂, 2.5 mM CaCl₂, 10 mM glucose, 10 mM Na-HEPES pH 7.25) containing 0.02% Pluronic-F27 (Invitrogen) for 15 min at 37°C, followed by washing in external solution for 15 min. Fluo4 and Rhod2 fluorescence were timelapse recorded (1 s intervals) with MetaMorph (Molecular Dynamics) on an Axiovert S100 microscope (Zeiss) equipped with appropriate filtersets (Chroma Technology), a 40x/1.3NA Plan-Neofluar objective (Zeiss) and a Photometrics Cascade-II 512B EMCCD. The cells were kept under constant perfusion with external solution (0.5 ml/min). IP3R-mediated Ca²⁺ release from ER stores was triggered by application of 100 μM Oxotremorine-M (Tocris) for 2 min. Ca²⁺ levels were calculated as relative Fluo4 or Rhod2 fluorescence compared to baseline fluorescence at the start of the measurement.