|
|
.
i. Indicator Form
We provide almost all of our indicators in two forms:
· Acetoxymethyl Form – This form, first introduced in 1981 by Dr. Tsien1,7, is
non-invasive and is technically the most popular method for loading fluorescent
calcium indicators into cells. The phenolic and carboxylic acid functions on
the molecule are derivatised as acetoxymethyl esters. These esters make the
molecule hydrophobic enough to be permeable to membranes, but they block its
binding to ions. Once inside the cell, non-specific esterases, found in almost
all cell types, hydrolyse the esters back to the polyanionic form necessary for
binding to calcium. Note: Dr. Tsien chose acetoxymethyl over a multitude of
esters based on the ease of hydrolysis. Pivaloylmethyl esters are currently
being promoted because of the ease of synthesis and they may be equally
effective.
· Potassium Salt Form – This form of the dye is soluble in water and is
available for calibration purposes to determine the dissociation constant of
the dye, for microinjection into cells,10 and for infusion into patch-clamps.9
It is normally sold in 1 mg aliquots but can be broken down into smaller
aliquots if required.
ii. AM Ester Loading Guidelines:
TEFLABS sells all AM forms of dyes in quantities of 1 mg, 500 μg, 250 µg,
20 x 50 µg, or 10 x 50 µg. We also offer them in dry DMSO solution (under
argon) and provide Pluronic, a surfactant that aids in dispersing the dye in
the aqueous buffer if needed.
For loading, a 1-10 mM stock solution is prepared using dry (anhydrous)
dimethylsulfoxide and kept at -20ºC. In order to prevent the deterioration of
the AM esters that occurs with repeated thawing and freezing, we recommend that
1 mg or 500 μg quantities be divided into aliquots containing 50 μg
each. Loading is usually performed in a serum-free culture medium at a
concentration ranging from 1-10 µM. Pluronic may be added to the loading medium
if there is difficulty in loading. (Not all cells require Pluronic.) The cells
can be incubated at 20ºC (or 37ºC), and the time of incubation may range from
30 to 60 minutes. They should be washed two to three times with fresh
serum-free culture medium for clean results. Note that incubation conditions
may vary from cell to cell and should be optimized for each cell type.
Protocols abound in the literature for a variety of cells.
iii. Calibration and Other Information
Calibration is necessary to obtain the dissociation constant (Kd) of each dye.
The dissociation constant factors in the calculation of ion concentration from
the fluorescence signal of the dye. Temperature, pH, ionic strength, and other
interactions of the dye in the environment such as proteins, viscosity,
etcetera will affect Kd. In fact, the Kd in the cell is typically higher than
the value obtained in vitro, so it is always important to determine it
accurately for the system under study.
The Kd of our calcium indicators varies, so it is important to select the dye
based on the concentration of calcium you expect from the experiment. In
general, the Kd is the midpoint of zero concentration to the saturation point
of calcium. Typical indicators show a dynamic range for calcium from 0.1 to 10
Kd.
There are generally two types of curves that are obtained when an ion binds to
these fluorescent calcium indicators. In one case, the resulting curve shows an
enhancement of fluorescence (Figure 1), whilst the other shows a spectral shift
in the emission or excitation spectrum (Figure 2). When there is an enhancement
of fluorescence, the resulting curves are either linearized by means of a Hill
plot or analyzed directly by non-linear regression to obtain the Kd20.
When there is a spectral shift in the emission or excitation, the calibration
involves a ratio of the fluorescence intensities measured at two wavelengths12.
The advantage of the ratiometric dye is that the ratioing of measurements
eliminates variable effects such as degree of cell loading, photo bleaching,
detector sensitivity, cell thickness and optical paths. It is indeed ideal, but
to date most ratiometric dyes are UV based.
|
