Biology Reference
In-Depth Information
as well as experimental procedures for, calibration for ratiometric and nonratio-
metric indicators are discussed in this section.
A. Calibrating a Nonratiometric Fluorescent Indicator
For a nonratiometric indicator that increases fluorescence emission on binding
Ca
2
þ
, the free Ca
2
þ
concentration is given by
¼ K
d
F F
min
F
max
F
Ca
2
þ
ð1Þ
where F
min
is the indicator fluorescence intensity at zero [Ca
2
þ
](whenall
indicator molecules in the sample are Ca
2
þ
-free), F
max
is the indicator fluores-
cence at saturatingly high [Ca
2
þ
] (when all indicator molecules are present as
the Ca
2
þ
-bound form), and F is the measured fluorescence intensity for which
we wish to find a corresponding value of [Ca
2
þ
]. To arrive at a correspondence
between measured F and [Ca
2
þ
]
i
, K
d
, F
min
,andF
max
all must be known.
Whereas K
d
usually is predetermined in vitro, F
min
and F
max
must be obtained
in situ. The most straightforward approach would be to try to equilibrate the
indicator-loaded cell with solutions that contain ''zero'' [Ca
2
þ
] and then high
[Ca
2
þ
]. In practice, however, deficiencies inherent in a nonratiometric indicator
make this approach unattractive. Interpretation of intensity changes is con-
founded by dye leakage, which causes the total indicator fluorescence from the
cell (and therefore F
min
and F
max
) to decrease with time. This basic flaw of
nonratiometric indicators means that obtaining good quantitative estimates of
[Ca
2
þ
]
i
in cells in which dye leakage or extrusion occurs at significant rates
would be di
cult. In such cases, a laborious calibration would not be justified.
An alternative semiquantitative calibration procedure developed for Fluo-3 is
discussed next.
The calibration procedure for Fluo-3 depends on the fact that,
Y
in vitro,
F
Mn
¼
0.2F
max
, where F
Mn
is the fluorescence intensity when Fluo-3 is saturated
completely with Mn
2
þ
(
Kao et al., 1989; Minta et al., 1989
). That F
max
¼
100F
min
is
also known from in vitro measurements. Because both F
max
and F
min
can be
expressed in terms of F
Mn
, the only parameter that must be determined experimen-
tally is F
Mn
. In situ calibration then consists of:
1. applying micromolar levels of ionomycin or Br-A23187 to increase perme-
ability of the cell to divalent metal ions;
2. adding su
cient MnCl
2
(typically twice the concentration of Ca
2
þ
in solu-
tion)
21
to ensure saturation of intracellular Fluo-3; and
Y
21
In Step 2, it is best if the medium contains no carbonate, bicarbonate, or phosphates, which can
form insoluble precipitates with Mn
2
þ
and thus reduce the concentration of free Mn
2
þ
. Moreover, the
light scattering by the particulate precipitates can add considerable noise to the fluorescence signal.
