Lsm 510 live microscope

HEK cells expressing AT₁R and Gαq-Gβγ FRET sensor were exposed to 1 nM AngII with or without 3 min pretreatment of 100 μM nifedipine. FRET was measured using a Zeiss LSM 510 LIVE microscope with a 40×/1.2 water immersion objective and excitation of 405 nm. YFP signal was corrected for mTurquoise bleedthrough and used as FRET intensity. Transfecting HEK cells with GαqmTq allowed for determination of donor bleed through into FRET channel. Bleed through coefficient, b, was calculated as I_mTq/I_FRET. In experiments, FRET channel intensities were then corrected as: FRET_corrected = I_FRET – b*I_mTq. FRET ratios were finally calculated as I_mTurquoise / I_{FRETcorrected}. Increase in FRET ratio corresponds with increased G_q-protein dissociation.

Intracellular calcium concentration ([Ca²⁺]i) in INS-1D cells was measured as previously described^{41 (link)} with a Calcium Fluo-4 kit (#CS22; Dojindo Molecular Technologies, Inc., Kumamoto, Japan). INS-1D cells were placed on a φ35 mm glass bottom culture dish, the growth medium was removed and replaced with loading buffer containing 3 mM glucose and Fluo4-AM, and the culture dish was incubated at 37 °C for 1 h. Loading buffer was removed and replaced with recording buffer. [Ca²⁺]i levels were detected when stimulated with 25 mM glucose and 30 mM KCl. Fluo-4 fluorescent signal over the INS-1D cell area was detected by excitation at 488 nm on an LSM 510 Live microscope (Carl Zeiss, Oberkochen, Germany).