Biology Reference
In-Depth Information
2. increasing extracellular [Ca
2
þ
] in the presence of Ca
2
þ
ionophore so that
Ca
2
þ
could enter the cell to saturate intracellular indicator;
3. permeabilizing the cell with digitonin (at concentrations prescribed in
Section III.B.2
) to release cytosolic dye so the background signal may be
measured.
23
Although the procedure seems straightforward, a few empirical findings are
helpful in performing a successful calibration:
1. Many cell types do not tolerate severe calcium deprivation well. During Step
1, these cells often become fragile or leaky or, in the case of adherent cells, detach
from the substrate. In many cases, however, one can compensate for the total
absence of Ca
2
þ
by supplementation with elevated concentrations of Mg
2
þ
. Thus,
raising the extracellular [Mg
2
þ
] to 5-20 mM can help maintain cell integrity during
a long calibration. Although Mg
2
þ
should bind Fura-2 to a limited extent and
slightly alter its fluorescence spectrum (
Grynkiewicz et al., 1985
), in practice R
min
is
not a
ected significantly by Mg
2
þ
supplementation.
2. Because ionomycin and Br-A23187 become very ine
V
cient at Ca
2
þ
transport
when intra- and extracellular free Ca
2
þ
concentrations are below micromolar
levels, depleting the cell of Ca
2
þ
entirely is quite di
Y
cult. Therefore, one often
must wait a long time for true R
min
to be reached in Step 1. In the example shown in
Fig. 11
, the interval between ionomycin addition and attainment of R
min
was in
excess of 90 min.
3. Step 2 could be performed in two ways. One could add, in combination with
fresh ionophore if desired, su
Y
cient Ca
2
þ
to bind to all the EGTA or BAPTA that
was introduced in Step 1 and still have a large excess of free extracellular Ca
2
þ
,as
well as su
Y
cient TRIS base to counteract any acidification arising from the Ca
2
þ
-
EGTA binding reaction. Alternatively, one could replace the medium from Step 1
with nominally Ca
2
þ
-free medium and then add a large excess of Ca
2
þ
in combi-
nation with a fresh dose of ionophore. The aim is to initiate massive Ca
2
þ
influx
into the cell at a rate that overcomes any Ca
2
þ
extrusion mechanism that the cell
may mobilize. In practice, concentrations of ionophore ranging from 10
6
to
10
5
M and external [Ca
2
þ
] in the range of one to several tens of millimolar can
be used.
Y
23
At controlled concentrations, digitonin releases primarily cytosolic dye whereas compartmenta-
lized dye remains with the permeabilized cell and would be subtracted out as background. The
assumption is that intraorganellar [Ca
2
þ
] is significantly higher (
> m
M) than cytosolic [Ca
2
þ
], so
compartmentalized indicator would be essentially completely Ca
2
þ
-bound and thus contribute a con-
stant background to the measured 340- and 380-nm fluorescence signals from the cell. This assumption
would fail if significant amounts of dye are compartmentalized into organelles that do not have high
luminal [Ca
2
þ
]. An alternative approach to obtaining a background reading is to add MnCl
2
(at a
concentration equal to or greater than the Ca
2
þ
concentration in the medium) at the same time as the
digitonin so that compartmentalized dye can also be quenched as ionophores transport Mn
2
þ
into the
organelles. Using such an approach assumes that cellular autofluorescence is the true background and
ignores the contribution of compartmentalized dye to the background.
