i am not able to understand bellow mentioned protocol, should i homogenize tissue then to florescence.
The intracellular free Ca2+ level in thoracic aorta during contraction was
measured based on the method described by Ozaki et al. (1991). The thoracic aorta
without endothelium was cut into segments approximately 8 mm in length and
1 mm in width under a dissecting microscope. After loaded with 10 mM of fura-
2/AM and 0.02% cremophor EL in a physiological salt solution (PSS; 136.9 mM
NaCl, 5.4 mM KCl, 1.0 mM MgCl2, 1.5 mM CaCl2, 23.8 mM NaHCO3, 0.01 mM EDTA,
and 5.5 mM glucose, pH 7.4) for 4 h, aortic strips were held horizontally in the
organ chamber of a fluorimeter (CAF-110; Jasco, Japan) filled with PSS. After the
aortic strips were pretreated with MMAIII for 1 h, PE was added to elicit
contraction. We measured the fluorescence intensity with 340 nm excitation
(F340) and 380 nm (F380) excitation. The ratio of F340 to F380 was calculated for
intracellular Ca2+ levels.
No. You need to keep to aorta strip/ring intact.The homogeneization of the tissue will destroy all the cellular components responsible for calcium homeostasis. The protocol above is missing an important step: the calibration of the signal. Furthermore, this method won't give you a direct measurement of calcium concentration. You can determine calcium concentration by using the formula developed by Grickiewitz et al. 1985. My advice would be to try to get training in a lab using such equipment as this is a tricky method.
i am facing problem the flourimeter is in other lab. and organ bath is automatic in my own lab.
so i am confused that after exposing tissue to the dye , intact ring can be put into the cuvette and enter the range for measuring fluorescence.
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