Læknablaðið : fylgirit - 01.05.1978, Page 151

seconds in a solution of 4C% formalin and concen-
trated (967o) etanol 1:9, washed gently in tap water
and incubated for 60 seconds in a mixture which
is made up of 100 ml 3C$> etanol, 0.3g benzidine
dihydrochloride, 1,0 ml 0.132 (3.87o) Zn SO4
7H2O, 1,0 g sodium acetat (NaC2H3023H20),
0.7 ml 37o H2O2, 0.2 g safranin, washed in tap
water and air dried. These reactions give red
cell nuclei and blue peroxidase positive structures.
C. The Leder technique (15). Air
dried cytocentrifuge preparations were fixed in
methanol formalin (conc.) 1:9 for 2-3 seconds
washed, dried and incubated in a mixture of 200
mg benzidine, 2 ml acetone, 4 ml dimetylsul-
phaxide, 32 ml.dest. water, and 0.08 ml 3%
H2O2. The mixture was filtrated before use.
During the incubation period the solution is kept
in a permanent movement. The preparations
are counterstained for 15 mins. in Mayer's
haemalum. These reactions give blue cell nuclei
and brownish to black peroxidase positive
structures.
Fractionation of mononuclear
leukocytes on nylon fibre columns
was performed essentially as described before
(24). Not more than 1.5 x 108 cells were
suspended in 15 ml of medium 199 with 207o heat
inactivated foetal calf serum (FCS) put on 30 ml
glass column containing 3.5 g nylon fibres
(Fenwal Lab. Morten Grave I 11) and incubated
for 20 minutes at 37°C. The cells were eluted
with 30 ml of culture medium 199 spun down
and suspended in the same medium.
Identification and depletion of Fc-
receptor bearing lymphocytes (EA-RFC)
Lymphocytes bearing Fc-receptors for IgG were
detected by using a rosette technique previously
reported (10,11,12,16) Human ORH+ erythrocytes
were sensitized with anti CD antiserum (Ripley).
Equal volumes of 1% erythrocyte suspension and
a cell suspension containing 1 x 106 cells/nil
were mixed, centrifuged for 2 minutes at 250 g
and left at room temperature for 15-20 minutes.
The cells were then gently resuspended and the
percenlage of EA-rosettes determined using a
Burker counting chamber. Lymphocytes with
five or more erythrocytes firmly attached to
their surface were difined as a rosette. After
counting the rosettes, cytocentrifuge preparations
were made and stained for peroxidase with modi-
fication of the Graham Knoll technique.
Depletion of EA-RFC was performed by EA-
rosette centrifugation (10,12,16). Briefly, the
lymphoid cell suspension were adjusted to 20 x
106/ml mixed with an equal volume of 5%
suspension of sensitized erythrocytes. After
centrifugation and incubation- the rosette con-
taining cell mixture was centrifuged on Isopaque-
Ficoll, the cells remaining in the interphace
were pipetted off and washed as described above.
Cytotoxicity assay. Antibody-dependent
cell mediated cytotaxicity (ADCC) was measured
as described before (24,25). For these experi-
ments, 2.5 x 10® mononuclear leukocytes in 0. 5
ml medium with antibiotics were mixed with
1 x 105 51 cr labelled chicken red cells (target
cells), 0. 5 ml of a 1:3 x 106 dilution of rabbit
antichicken eruthrocyte antiserum and 0.05 ml
of heat inactivated foetal calf serum. Medium
was used instead of antiserum in the control
cultures. All experiments were performed in
duplicate. Tubes were incubated at 37° for
24 hours in an atmosphere of 97o CO2 and 10Ö7o
humidity. They were then centrifuged at 150 g
for 10 minutes. The total radioactivity of each
tube and the radioactivity of 1.0 ml of the super-
natant were measured in a gamma ray counter.
(N. E. 8312 Nuclear Enterprise Edinburgh). For
each tube the percentage release was expressed
as cytotoxic index (CI).
_T _ radioactivity of the supernatant .
total radioactivity
Lymphocyte cultures with mitogens
Isopaque/Ficoll separated mononuclear leukocytes
were cultured with pokeweed mitogen (PWM)
phytohemagglutinin (PHA) concanavalin A (Con A),
both before and after fractionation on a nylon
fibre column. The proportion of EA-RFC and
the percentage peroxidase-positive cells was
determined before as well as after 3 days culture.
The cultured cells were washed thoroughly in
Hanks BSS before the tests were performed. The
lymphocyte culture technique has been described
in detail earlier (8,9).
R esults:
Peroxidase-pos i t iv e cells separated
by the Isopaque-Ficoll gradient
c entrifu gation
Table I shows the results of experiments with
three different peroxidase staining methods on
Isopaque-Ficoll gradient separated lymphocytes
from 14 different individuals. The peroxidase
technique of Leder gave the lowest means (14,7%)
and range (12.1-18.97o). The two other techniques
gave practically the same results with means of
20.77o and 20.87o, respectively (Table 1). The
Kruskal-WaUis rank test gave a quite good corre-
lation between the three methods. (21) (Hí 4.921
p>0.05). The peroxidase negative cells with
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