WPI SI-BF-100, Инструкция по эксплуатации

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SI-BF-100
World Precision Instruments 19
APPENDIX A: EXAMPLES OF CALCIUM
MEASUREMENT
Intracellular Calcium Concentration Measurement
When muscles are stimulated by the neurotransmitters released from motor
nerves, muscle fi bers develop action potentials that spread into the depths of the
muscle through a network of tubules. These action potentials alter the membrane
potentials of organelles inside the fi bers and cause the release of calcium from the
sarcoplasmic reticulum (SR). The calcium released from the sarcoplasmic reticulum
is required for the binding of myosin and actin, which are the molecules inside the
muscle fi bers that are responsible for contraction.
1. Calcium attaches itself to the actin fi lament and causes the binding site for
myosin to open and be available for the head of the myosin molecule to bind to
the actin fi lament.
2. Once attached, the head of the myosin molecule pulls the actin fi lament toward
the center of the functional unit of the muscle fi ber, the sarcomere.
3. As the sarcomere shortens, the muscle fi ber and the muscle shorten.
4. When a defi ciency in the concentration of calcium in the fl uid around the
muscle fi bers exists, the myosin binding sites on the actin fi laments are covered
and the fi ber contraction stops.
The intracellular concentration of calcium can be determined by measuring the
fl uorescence of an indicator dye, like Fluo-4, Indo-1, or Fura-2, that binds to calcium
inside the cells.
1. The lipid-soluble, acetoxymethyl (AM) ester form of any of these dyes can be
loaded into intact muscle fi bers using non-invasive techniques.
2. Once inside the cells, the ester portion is cleaved from the molecule and the dye
segment is free to bind to calcium.
3. When the dye binds to calcium, the dye fl uoresces. Since the dye is no longer
lipid-soluble, it is trapped inside the fi ber. Therefore, any fl uorescence, which is
measured by the
SI-BF-100 , is indicative of the amount of calcium inside the
fi bers.
When confi gured for calcium measurements with a particular dye or combination of
dyes, the
SI-BF-100 Biofl uorometer is supplied with all the components needed to
measure calcium concentrations in intact muscles and fi bers. When combined with a
SI-MKB muscle tester, the force of contraction from muscle fi bers can be measured
at the same time that calcium transients are recorded.
Calcium Measurements with Fluo-4
Fluo-4 is a commercially-available, single-excitation, single-emission calcium
fl uorophore that can be used to make real-time calcium measurements from tissue.