
2 A Rich and A. K. Harris
reverse contact inhibition relative to fibroblasts (Harris, 1974). We have found that
macrophages also show significant differences in their adhesive preferences.
MATERIALS
AND
METHODS
Peritoneal macrophages were obtained from Swiss white mice, which were killed
by
cervical
dislocation
and the
cells collected
in 3-4
ml
of
phosphate buffered saline (PBS) (Dulbecco
&
Vogt, 1954).
In
some experiments, heparin was included
in the
saline,
but
seemed
to
make
no
difference
to the
results. After centrifugation,
the
cells were resuspended
in
PBS
and
allowed
to settle onto
the
experimental substrata
for
15-20 min
at
37 °C, before replacing
the
saline
with Dulbecco's modified Eagles medium buffered with 25 mM-.N-2-hydroxyethylpiperazine-
N'-ethanesulfonic acid (HEPES),
and
supplemented with
10%
foetal calf serum (GIBCO,
Grand Island, N.Y.)
and
penicillin
and
streptomycin.
For
certain experiments, 5
ml of
thio-
glycollate medium (Fisher Scientific Co.) was injected intraperitoneally 5 days prior
to
collect-
ing macrophages
as
above.
A
culture
of
the macrophage-like cell line, P338D,, was
a
gift from
Dr H.
S.
Koren, Duke University. WI38 cells, VA13 cells (a line
of
untransformed fibroblasts),
simian virus-transformed VA13 cells,
and
L929 cells
(a
transformed line
of
mouse fibroblasts)
were
all
obtained from
the
Tissue Culture Laboratory, Cancer Research Center, University
of
North Carolina
at
Chapel Hill.
H615 ts 3T3
cells were
a
generous gift from
Dr
Kenneth
Noonan
of
the University
of
Florida.
Three artificial substrata were used:
(1)
untreated polystyrene dishes, 35
mm in
diameter;
(2) polystyrene dishes treated with concentrated sulphuric acid
for
2
h to
make
the
substratum
hydrophilic (Maroudas, 1975);
and (3)
palladium evaporated onto treated
and
untreated poly-
styrene (Carter, 1967ft).
All 3 of
these culture surfaces could
be
produced side-by-side
in
individual Petri dishes. Approximately 2
x io
5
macrophages were plated
on
1 dish.
Slightly roughened regions were created
on
Falconized polystyrene surfaces
of
tissue culture
flasks by gently sliding
the
round, fire-polished
end of
a
glass
rod
across
the
surface so that
the
rough areas were just barely visible
by
phase-contract microscopy. Approximately 2
x io
6
cells
were plated
per
25 cm* flask.
Cultures were viewed with
an
Olympus
CK
inverted phase-contrast microscope
and
photo-
graphed with
an
Olympus 35
mm
camera using Kodak Panatomic
X
film. Individual frames
from
a
16 mm Bolex camera using Plus
X
reversal film were converted
to
120 film
by
a Testrite
cine-enlarger. During observations,
a
Sage air-current incubator maintained
the
temperature
of the cultures
at
37
C
C.
RESULTS
When plated out on grid patterns of alternative substrata, both the transformed
(VA13 and L929) and untransformed (WI38 and ts 3T3) fibroblasts migrated pre-
ferentially onto the more hydrophilic material available to them (i.e. untreated poly-
styrene < palladium < sulphonated polystyrene). Peritoneal macrophages, however,
developed the opposite sequence of relative preference to these 3 substrata.
Macrophages accumulated on untreated polystyrene in preference to palladium
and on palladium in preference to sulphonated polystyrene (Fig.
1
c,
D).
In addition,
a greater percentage of macrophages flattened themselves onto the more preferred
substrata. This reversed pattern of accumulation occurred when foetal calf serum or
beef heart infusion was used to supplement the medium.
Another unusual characteristic of macrophages is the length of time required for
these reversed-substratum preferences to develop. Fibroblasts accumulated on their
preferred substrata within 1 day but macrophages required between 1 and 2 days
相关文档
评论