This protocol is significant because it uses patient material. If the patient has the disease, using their cells should more accurately reflect the patient's biological response. This technique is simple to perform, straightforward, and does not require sophisticated equipment.
These methods can be used for diagnosis, for example, to prove that a RYR1 mutation functionally changes calcium homeostasis, as well as to test the potential therapeutic function of a compound. Depending on the knowledge and technical skills of the individual researcher, test experiments should be performed before actual patient samples are used. To monitor changes in the intracellular calcium concentration of EBV-transformed B lymphocytes, resuspend the immortalized B cells at a one times 10 to the seventh cells per milliliter concentration in Krebs Ringer's solution and Fura-2 AM to a final concentration of five micromolar for a 30-minute incubation at 37 degrees Celsius.
At the end of the incubation, collect the cells by centrifugation, and resuspend the pellet in fresh Krebs Ringer's solution at a two times 10 to the sixth cells per milliliter concentration. Just before starting the experiment, centrifuge the cells again, and quickly resuspend the cells in 1.5 milliliters of Krebs Ringer's solution supplemented with 0.5-millimolar EGTA but no added calcium. Transfer the cells to a glass, three-milliliter spectrofluorometer cuvette, and record the fluorescence ratio on a spectrofluorometer equipped with a magnetic stirrer set at maximum velocity and 37 degrees Celsius for about 30 seconds.
To assess the calcium release response in single cells, plate one times 106 Fura-2 AM loaded cells in one milliliter of Krebs Ringer's solution supplemented with one-millimolar calcium chloride onto individual poly-L-lysine coated glass cover slips, and incubate the cover slips at 37 degrees Celsius in a humidified cell culture incubator for 30 minutes. When the cells have attached, place a cover slip into a perfusion chamber, and begin perfusing the cells at a two milliliters of Krebs Ringer's solution supplemented with one-millimolar calcium per minute flow rate. Using an inverted fluorescent microscope equipped with a 40 times oil immersion objective and the appropriate filters, record online measurements with a software-controlled, charge-coupled device camera attachment at one-second intervals, at a fixed exposure time.
To achieve cell stimulation, use a cell perfusion stimulator with 12 valves to add the different concentrations of agonist. To isolate myotubes from human muscle biopsies, first rinse the biopsy with sterile PBS to remove any excess blood before mincing the tissue into small, 0.5 to one millimeter fragments. Place two to three fragments into individual inserts in each well of a six-well tissue culture plate containing 1.5 milliliters of human muscle growth medium per well and 500 microliters of medium per insert, and place the plate into the cell culture incubator.
To measure changes in calcium expression in human myotubes, when multinucleated myotubes can be observed, replace the myotube culture supernatants with fresh differentiation medium supplemented with 10 microliters of one-millimolar Fura-2 AM for a 30-minute incubation in the cell culture incubator. At the end of the incubation, transfer one glass cover slip culture to the perfusion chamber, and rinse the cells with Krebs Ringer's solution supplemented with two-millimolar calcium chloride. Then perform online calcium measurements as demonstrated using a 20 times water immersion objective.
In this representative analysis, a rapid increase in calcium was observed in EBV-immortalized B lymphocytes after the addition of agonist that slowly declined back to resting levels over time. In a similar experiment, the addition of 400-nanomolar thapsigargin caused a large calcium transient that reached a peak fluorescence value of 2.4 arbitrary units. This fluorescence value was considered to be 100%when the corresponding dose response curve was constructed.
In this analysis, immortalized B cells were stimulated with five-millimolar caffeine for 20 seconds, resulting in an immediate increase in the 340 and 380-nanometer fluorescence ratio. For each caffeine concentration tested, the caffeine-induced peak at the ratio of 340 by 380 nanometers of fluorescence was calculated and used to construct a dose response curve. Calcium release from differentiated myotubes can also be assessed in response to flushing with potassium chloride, different concentrations of agonist, and/or caffeine, and normal dose response curves can be generated.
It is important to make sure that the cells are loaded properly with Fura-2 and that both the 340 and 380-nanometer wavelengths respond to the addition of the agonist. Intracellular calcium measurements with fluorescent calcium indicators can be investigated in most mammalian cells and can be applied to the study of intracellular calcium changes in response to different stimuli.
