Biology Reference
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lene, which may or may not be coated with single ECM molecules, mixtures of
molecules, complex matrices such as Matrigel and Humatrix (39) , cell mono-
layers, or reconstituted tissues and tissue slices.
1.4. Assessing Transfilter Cell Migration
Scoring chemotaxis, random and directed cell movement, and invasion
through the porous membranes of Transwells has conventionally relied upon
visual counting of stained cells (37 , 38) . Obviously, visual counting of cells
dispersed over a membrane is subjective and time-consuming. Furthermore, it
is virtually impossible to determine the true percentage of cell migration and/
or invasion because of the difficulties in scoring the total cell number con-
tained in the system by simple visual counting. In order to be able to accom-
plish a more precise cell counting, colorimetric procedures that are based on
the use of dyes, such as toluidine blue (38) , or cell viability markers, such as
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenol tetrazolium bromide (MTT; ref. 40 )
have been adopted. After dye incorporation, cells are solubilized and the
detected absorbance is directly correlated with the number of cells attached to
the underside of the membrane (i.e., transmigrated cells). Major drawbacks
with these colorimetric methods are the potential of a variable background
staining associated with protein-coated membranes; the variable toluidine blue
staining of different cell types; and the extra incubation times needed to
accomplish the procedure. Furthermore, if the migratory capacity of leuko-
cytes is studied, additional steps may be needed to accomplish the proce-
dure. For instance, it may be necessary to centrifuge the plate to allow the
transmigrated leukocytes to more firmly adhere to the bottom of the well, such
as to permit the removal of the culture medium and the addition of the suitable
dye solution.
Isotope-based assays provide an alluring alternative to the rather unprecise
colorimetric procedures because they are sensitive and afford an efficient
means of quantitating the number of transmigrated cells (37 , 38) . However,
apart from the inconvenient and hazardous nature of isotopes, as well as the
time-consuming step of prelabeling cells metabolically, an additional problem
is that associated with the fact that migration assays are long-term assays that
may be protracted for days. Thus, the spontaneous release of radioactivity by
the moving cells may be the cause of overestimations of the actual number of
migratory/invasive cells. It has more recently been realized that this problem
can be circumvented by employing fluorescent cell tagging, via calcein-AM
(34 , 35 , 41) or BCECF-AM (35 , 41) . A suitable protocol to follow in this case is
to utilize the fluorochrome calcein AM to label by spontaneous uptake those
cells that have transversed the porous membrane of the Transwell. However,
this labeling step has to be preceded by removal of the nonmigrated/invading
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