April - June 2007 - Kasetsart University

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medium containing 100 µg/ml ampicillin, 25 µg/
ml kanamycin and 1 mM isopropyl-1-1-thio-β-Dgalactoside
(IPTG). Cells were harvested and
extracted by denaturing condition and then the
recombinant IL-2 was purified with Ni-NTA resin
affinity column chromatography according to the
recombinant protein purification procedures
(Qiagen, Valencia, USA). The rmIL-2 was allowed
to refold in native conformation by dialysis in PBS
and the protein concentration was determined by
Bradford protein assay (Bradford, 1976). The
protein purity was determined by SDS-PAGE
(Laemmli, 1970).
Western blotting
Twenty micrograms of the rmIL-2 was
loaded in a mini-gel apparatus and resolved on a
12% SDS-PAGE gel and transferred to
nitrocellulose membrane by electroblotter (Bio-
Rad, California, USA). The blot was blocked with
5% skim milk, incubated with rat anti-mouse
interleukin 2 IgG monoclonal antibody (Serotech,
North Carolina, USA) (1 µg/ml) for 30 min at
room temperature. After washing, it was incubated
with goat anti-rat IgG conjugated with alkaline
phosphatase at 1:10,000 dilution (Sigma-Aldrich,
St. Louis, USA). Detection was performed using
the 5-bromo-4-chloro-3-indolyl phosphate/
nitroblue tetrazolium (Zymed, South San Fancisco,
USA) as substrates.
Cell proliferation assay
The rmIL-2 was investigated for the
ability to stimulate cell proliferation which was
quantified by the colorimetric assay based on the
2,3-bis (2-Methoxy-4-nitro-5-sulfophenyl)-5-
[(phenylamino)-carbonyl]-2H-tetrazolium
hydroxide (XTT) assay as previously described
(Scudiero et al., 1988).
Splenocytes were transferred into 96 well
microtitre plates (Costar, New York, USA) at a
density of 1 × 10 5 cells/well for 48 h in complete
medium. The XTT (Sigma-Aldrich, St. Louis,
Kasetsart J. (Nat. Sci.) 41(2)
USA) solution was prepared freshly at 1 mg/ml in
prewarmed balance salt solution without phenol
red. Then, 5 mM phenazine methosulfate (PMS)
(Sigma-Aldrich, St. Louis, USA) solution was
prepared in PBS, stored at 4°C until use and
protected from light. Culture medium was
removed from each well, after that a 50 µl of XTT
solution with 0.025 mM phenazine methosulfate
was added. After 5 h of incubation, the absorbance
at 450 nm was determined by a Multiskan EX
(Labsystems, Finland).
Receptor binding assay
A New Zealand white rabbit was first
immunized with a mixture of rmIL-2 (1 mg/ml)
and Freund’s Complete adjuvant (Sigma-Aldrich,
St. Louis, USA) at 1:1 ratio following by three
injections at weekly intervals with the same
antigen and Freund’s Incomplete adjuvant (Sigma-
Aldrich, St. Louis, USA). The antiserum with the
highest titre was used for immunofluorescent
detection of IL-2 receptor binding. The splenocytes
were stimulated with Con A mitogen at 5 µg/ml
final concentration compared with non-stimulated
control. Cells were incubated for 6 h at 37°C in
5% CO 2 and harvested for receptor binding assay.
They were washed twice with PBS and then
resuspended in 50 µl of PBS, and fixed with 100
µl of 4% paraformaldehyde for 30 min in the dark
at 4°C. The experiment was done on a glass slide.
Proteins on cell surface were stained with 500 µM
sulforhodamine B (SRB) (Sigma-Aldrich, St.
Louis, USA), washed with 1% acetic acid and
PBS. Cells were then incubated with recombinant
IL-2 for 1 h at 37°C, washed with PBS and reacted
with rabbit anti-rmIL-2 polyclonal antibody
(1:500). After washing step, FITC goat anti-rabbit
IgG (H+L) conjugate (Zymed, South San
Fancisco, USA) (1:50) was added and incubated
for 1 h at 37°C and then washed with PBS. Cells
were analyzed by reflected light fluorescence
illuminator BH2-RFL (Olympus, New York, USA)
within 5 h.