Biology Reference
In-Depth Information
ferred over native BSA for most cell types. Our experience, however, and that
reported by others, indicate that this may not always be the case. Alternative
blocking agents to consider may then be human serum albumin,
-casein and
ovalbumin, in their native or denatured form. Avian neural crest cells, for
instance, bind to some extent to BSA
(5
,
6)
, but not to ovalbumin, whereas human
and murine lymphocytic cells may bind to
α
-casein and ovalbumin, but fail to
bind to denatured BSA. On the other hand, fibroblastic cells such as the human
rhabdomyosarcoma RD-KD and the human embryonic kidney 293 cells bind to
both native BSA and ovalbumin, but do not interact with
α
-casein and denatured
BSA. Thus, it may be necessary to identify empirically the suitable blocking
agent for each given cell type. In this context the most difficult situation that we
have faced has been that of B lymphocytes freshly isolated from patients affected
by chronic lymphocytic B cell leukemia or adult myeloid leukemia. In these cases,
we have found that 1% human serum albumin plus 0.5% Tween-20, was the sole
saturating agent that consistently yielded a low background binding. Finally,
we find that when the adhesion assays are run in the presence of the stimulating
Mn
2+
divalent cation, or activating anti-integrin antibodies, the nonspecific inter-
action of the cells with these blocking proteins has a tendency to be exaggerat-
edly enhanced. Caution should be taken when carrying out cell-adhesion assays
under these conditions.
α
4.
Use wells filled only with blocking agent as negative control. We normally adopt
a “background” cell binding of <10% for these control wells as threshold value
for judging that the experiment was successful.
5.
Calcein AM is a colorless polyanionic fluorescein derivative that, upon digestion
by cytoplasmic esterases becomes fluorescent (
λ
em
535). The cleavage
of the AM group by acetylases causes the calcein molecule to become negatively
charged and prevents it from rapidly diffusing out of the cells. In fact, in com-
parison with the thiol-reactive fluorescent dyes CellTrackers (Molecular Probes,
Inc.), which can alternatively be used as vital cell tracers, we find that calcein
AM exhibits higher retention time. On the other hand CellTrackers generally
produce a somewhat more intense cell labeling and can effectively be used for
multiple cell labeling. Calcein AM is inoquous to the cells and is not known to
influence their adhesive behavior.
λ
ex
485,
6.
The function of the PVP is to provide a higher viscosity of the medium, such as to
match the relative density of the cells, and is an inert substitute to the previously
utilized BSA
(4-8)
. Thus, the exact concentration of PVP may vary depending
upon the cell type analyzed. The range of 0.1-0.5% is the one that, in most cases,
would satisfy the viscosity equilibrium requirement. For lymphocytes we nor-
mally use 0.1%, whereas for larger cells as fibroblasts, epithelial cells, endothe-
lial cells, and various tumor cells, we use 0.5%. When analyses of
cation-dependency of cell binding are to be carried out, we recommend using as
cell adhesion medium: 0.25 m
M
Tris-HCl, pH 7.4, with 0.15
M
NaCl to which
the different cations can be added at the desired concentrations. In this condition,
there is no detectable precipitation of cations, even when applied at rather high
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