Adherent Primary Cultures of Mouse Intercostal Muscle Fibers for Isolated Fiber Studies
Figure 4
Fluo4 transients elicited by field stimulation in intercostal and FDB fibers. Representative line-scan images of transient responses in FDB (a) and intercostal (b) fibers to a 5 sec 10 Hz train of field stimulation applied at time zero, performed with nonratiometric fluo-4 AM loaded fibers and assayed with high-speed confocal line scan microscopy (100 s/line). Images in top of panels (a) and (b) show xy images of FDB and intercostals fibers to indicate the location of the scan line, scale bar is 20 μm. Note that fluo-4 signal at rest in these fibers is very low. Traces under the images are the time courses of the fluo4 transients. The white lines in the time domain images indicate the region of interest drawn at the center of the fiber and used to calculate the change in fluorescence in response to field stimulation. Data presented as ΔF/F0. Fluorescence was detected with a Zeiss LSM 5 Live ultrafast confocal system based on an Axiovert 200M inverted microscope. Fibers were imaged with a 63x/NA 1.2 water immersion objective lens. Excitation for fluo-4 was provided by the 488 nm line of a 100 mW diode laser, and emitted light was collected at >510 nm.
