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International Journal of Marine Science 2013, Vol.3, No.19, 151-157
http://ijms.sophiapublisher.com
154
drained; the pellet was then resuspended in 300 µL of
RPMI 1640 medium.
Twelve to fourteen centimetre of drinking straw was
cut. One end of the straw was slantly cut and sealed by
slightly heating the tip in a flame. Nylon wool fibres
were finely teased using a pair of forceps and the teased
fibres were packed (loosely) into the straw. Adding 5
mL of physiological saline washed the packed nylon
wool column. A small opening was made at the sealed
end of the straw to drain the physiological saline. After
washing with physiological saline, the nylon wool was
then filled with 3 mL of RPMI 1640 medium in a
horizontal position. The nylon wool column was kept
in the incubator (at 37
℃
for 30 min) in horizontal
position. This process activates the nylon wool column.
Resuspended lymphocytes were loaded into the
activated nylon wool column then the column was held
vertically above an eppendrof tube, now hot saline
(about 60
℃
) was slowly dripped into the column. The
hot saline passing out of the column was collected in
the eppendrof tube, which contain and T lymphocytes.
After hot saline elution, cold saline in then dripped was
through the column. The column in gently squeezed to
release the adhered B cells (repeat twice). The cold
saline dripping out of the column was collected in
another eppendrof tube. 0.2 mL of the saline containing
B lymphocyte (from the eppendrof tube containing B
cell) was taken in a separate eppendrof tube. To this 0.2
mL of 1% SRBC was added and then the mixture was
centrifuged for 12 minutes at 1600 rpm. After
centrifugation the sample were incubated in an icebox
or refrigerator (at 4
℃
) for 5 minutes. After cold
incubation, the pellet in the eppendrof tube was re-
suspended by gentle flushing with a Pasteur pipette.
Then a drop of it was taken in a clean dry slide,
observed and enumerated B cells under the microscope
(20×/40×) for rosettes. Numbers of B cell rosettes
formed were observed among hundred lymphocytes
observed/mL was tabulated.
2.2.6 Delayed type hypersensitivity (DTH) response
in rats
On 14
th
day of the study, all the groups I to IV were
immunized with SRBCs (0.1mL of 20% SRBC i.p.) in
normal saline. On day 21
st
all animals from all the
groups were challenged with 0.03 mL of 20% SRBCs
in sub plantar region of right hind paw. Foot pad
edema in rat was used for detection of cellular
immune response. On 21
st
day, injection of 0.1 mL of
20% SRBCs in the sub plantar region of right hind
paw in the volume of 0.03 mL and normal saline in
left hind paw in same volume as well as the purified
compound (50 mg/kg, 150 mg/kg and 250 mg/kg)
doses was given. Foot pad reaction was assessed every
3, 6, 9, 12 and 24 hrs. On 22
nd
day, in terms of
increase in the thickness of footpad as a result of
hypersensitivity reaction due to edema, the thickness
of the right hind footpad was measured using vernier
caliper. The footpad reaction was expressed as the
difference in the thickness (mm) between the right
foot pad injected with SRBC and the left footpad
injected with normal saline.
2.2.7 T cell rosette assay
Methodology
Blood is collected from sponge extract treated, control
and standard drug treated rat as mentioned in earlier
section. T cell count in the blood is carried out by the
following method.
Five to ten ml of blood was collected and it was
introduced into sterile conical flask/beaker containing
(4~5) sterile glass beads. It was then continuously
swirled until no sounds were heard from the beads.
This indicates that all the fibrins have adhered to the
beads. This blood was considered as defibrinated blood.
This defibrinated blood was taken and diluted with
equal volume of physiological saline. 3 mL of the
lymphoprep solution was taken in a centrifuge tube.
The tube was kept in slanting position and 9 mL of
diluted blood was slowly added along the sides of the
centrifuge tube using Pasteur pipette. Care was taken
so that the FICON layer of the lymphoprep solution
present in the centrifuge tube was not disturbed. The
content of the centrifuge tube was then centrifuged at
1600 rpm for 20 min. The interphase (containing
lymphocytes) was removed using pipette. The cells
were washed with 1 mL saline and excess FICON was
removed. The sample was again washed with 1 mL of
saline after centrifugation the supernatant was decanted
by inverting the tube over a filter paper after all saline
was drained; the pellet was then resuspended in 300 µL
of RPMI 1640 medium.
Twelve to fourteen centimetre of drinking straw was
cut. One end of the straw was slantly cut and sealed by