Biology Reference
In-Depth Information
Probenecid and sulfinpyrazone are both hydrophobic organic acids and, as such, are
practically insoluble in water. These molecules must be neutralized with a stoichio-
metric amount of base (e.g., NaOH) before an aqueous stock solution can be
prepared.
D. Procedure for Loading
Stock solutions of the AM ester of the indicator can be made with dry DMSO as
solvent. Typical concentrations can be in the range of 0.1-10 mM. Such DMSO
solutions may be stored safely in screw-capped polypropylene microcentrifuge
tubes in the freezer for many months without apparent degradation. Dry DMSO
is used for making a solution of Pluronic F-127 at a concentration of 15-20% (w/w
or w/v is not crucial). This stock solution can be stored at room temperature. When
exposed to air, this concentrated stock solution slowly absorbs moisture until
Pluronic F-127 begins to precipitate. Because a heterogeneous Pluronic stock is
di
cult to transfer, preparing a fresh stock is preferable at this stage. Usually one
can load cells by incubation in medium containing (nominally) 0.1 to a few tens of
micromolar AM ester in a Pluronic dispersion. Typically 0.5-1
m
L of Pluronic
stock is su
Y
cient for dispersing 1-10 nmol AM ester. Thus, 1
m
L of 20% Pluronic
stock is adequate for dispersing 1
m
L of 10 mM AM ester stock into 1 mL of
medium to yield a 10
m
M AM ester solution (1000-fold dilution). Using a dilution
of
Y
1:1000 minimizes the DMSO concentration in the final loading medium (to a
few parts per thousand). Premixing the requisite volumes of AM ester stock and
Pluronic stock is advisable since this minimizes the chances of AM ester precipita-
tion during aqueous dispersal. The Pluronic-AM mixture is then dispersed into
aqueous loading medium.
Serum proteins [e.g., bovine serum albumin (BSA)] often have a salutary e
V
ect
Y
on cells and also can improve loading e
ciency, possibly by acting as hydrophobic
carriers for the AM ester and preventing its precipitation. Thus, including BSA
(0.5-1%) or serum (a small percentage) in the incubation medium may be
advantageous.
Enzymatic processing of an AM species to yield the Ca
2
þ
-sensitive indicator
typically consists of sequential hydrolysis of four to eight AM ester groups,
depending on the particular indicator chosen. Only when all the AM groups on a
molecule are hydrolyzed does the molecule become properly Ca
2
þ
-sensitive. For
this reason, after removal of AM-containing loading medium, allowing the cells
some extra time (e.g., 20 min at room temperature) to complete intracellular
processing of the most recently trapped, partially hydrolyzed AM species can be
useful in some cases.
It is reassuring to convince oneself that su
cient AM ester is present in the
loading medium, so the amount of indicator taken up by cells is not limited by the
amount available. For a typical example, assume that 1 million cells are loaded in
2 mL medium containing 2
m
M AM ester. The total amount of AM ester available
Y
