To begin, mount a clean glass slide on the experimental bath chamber. Coat the slide with two to three microliters of laminin and let it dry for 30 seconds. Then, pour approximately 400 microliters of the muscle fiber suspension onto the slide and allow the fibers to adhere to the laminin for 10 to 15 minutes.
Place the experimental chamber onto the stage of an inverted microscope equipped for epifluorescence. To verify the viability of the fibers, place two platinum electrodes on either side of the experimental chamber. After applying rectangular current pulses for 0.8 to 1.2 milliseconds, observe the muscle fiber contractions at the extremes.
Load the fibers with 3.5 to 4.5 micromolars of the fast calcium dye Mag-Fluo-4 AM for four to five minutes in Tyrode solution. After washing the slide with Tyrode solution, let the intracellular dye de-esterify for 15 to 20 minutes in the dark. During the acquisition of calcium transients under dimmed illumination, collect and save the light signals with an oil immersion 40X objective and a photomultiplier tube connected to a digitizer.
In the acquisition software, ensure a scale of zero to 200 arbitrary units. Then, adjust the size of the excitation spot and the gain of the photomultiplier tube to set the resting fluorescence, or F-rest, to 10 arbitrary units. For analyzing the recordings, set a low-pass filter to the whole trace at one kilohertz.
Then, adjust the peak to calculate the F-rest in one second of the trace. Adjust the F-rest to zero and measure the peak sarcoplasmic calcium transient's amplitude. Measure the rise time from 10%to 90%of the amplitude, the duration at half-maximum, and the decay time from 90%to 10%of the amplitude.
Then, estimate the decay kinetics according to a fit with the bi-exponential function. Calculate the peak calcium concentration in micromolar. Finally, save the values of the time constants of decay tau 1 and tau 2, and amplitudes A1 and A2.The FDB and EDL muscles showed calcium kinetics known as morphology type II Soleus muscles displayed morphology type I calcium transients.
Fibers from PDQA, PL, and EHL muscles shared the type II morphology. The calcium transient's kinetic signals of PDQA, PL, and EHL were similar to the signals of FDB EDL muscles, but differed from soleus muscle signals.
