Lsm 510 microscope

Cells were incubated with Alexa 488-CTB (Invitrogen) in the dark for 30 min on ice. Cells were briefly rinsed with ice-cold media and placed on ice in the dark until use. Imaging and photobleaching were conducted on a Zeiss LSM510 microscope (Carl Zeiss) equipped with an Ar 488 nm laser. 60 iterations at 100% of 488 nm excitation was used to photobleach a small portion (31 px diameter) of the membrane. Adjacent non photobleached membrane areas of the same size were used as a control for sequential bleaching as previously described (Tecedor et al., 2013 (link)).

Ca²⁺ transients and sarcoplasmic reticulum Ca²⁺ content were assessed in cardiac ventricular myocytes isolated from 12‐week‐old male MK2^+/+ and MK2^−/− mice as described previously.⁴³ Briefly, myocytes were incubated with 10 μmol/L Fluo‐4 AM (Molecular Probes, Carlsbad, CA), then transferred to a perfusion chamber on the stage of a Zeiss LSM 510 microscope (Carl Zeiss AG, Jena, Germany), and perfused with a Tyrode solution. The perfusion chamber was fitted with bipolar platinum electrodes attached to a Grass SD9 stimulator and maintained at 37°C. To assess Ca²⁺ transients, myocytes were continuously field stimulated at a rate of 2 Hz. To assess the sarcoplasmic reticulum Ca²⁺ content, myocytes first received 10 conditioning pulses at 2 Hz to ensure that each cell had a similar activation history. Upon completion of the conditioning pulses, 10 μmol/L caffeine was applied to the myocyte for 10 seconds via a rapid solution switcher. Changes in free Ca²⁺ were measured in line scan mode with excitation at 488 nm and emission measured at 505 to 530 nm. Myocytes were scanned repeatedly along the length of the cell at 1.5‐ms intervals for 7 seconds. Sequential scans were stacked to create a two‐dimensional image. Image J (National Institutes of Health, Bethesda, MD) was used to visualize the Ca²⁺ transients, and the data were analyzed with pCLAMP 8.2 (Molecular Devices).