Measuring Cytosolic and Mitochondrial Ca2+ Levels

Cytosolic and mitochondrial Ca²⁺ levels were measured following
IP3R-mediated release from ER stores in HEK293 cells20 (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 and
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 × 40/1.3NA 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⁻¹).
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 with
baseline fluorescence at the start of the measurement.