Preparation of Agaro-oligosaccharides Agar was resuspended in O.lN HCI to 10% (w/v) and heated at 100°C for 15 min. After centrifuging the sample to remove the insolubles, the Agaro-oligosaccharide solution was subjected to gel filtration chromatography in Toyopearl HW-40C (45 mm x 100 cm, Toso) to obtain Agaro-oligosaccharides of various lengths. The gel filtration chromatography was conducted at the rate of 1 mumin carrier. TLC and pure water was used as the The various fractions were analyzed by thin layer chromatography (TLC) using silica gel 60 F 254 and butanol : ethanol : water (5 : 3 : 3) as the solvent system. The analysis results indicated that various Agaro-oligosaccharide fractions were obtained, so that Agarobiose, Agarotetraose and Agarohexaose were prepared. Cell culture of RAW264.7 The mouse macrophage cell line RAW264.7 (ATCC TIB71) was obtained from s Dainippon Pharmaceutical Co., Ltd. The cells were cultured in Dulbecco’ modified Eagle’ medium (BioWhittaker) containing 10% fetal bovine serum (JRH Biosciences) s and antibiotics (50 units/ml penicillin, 50 pg/mL streptomycin, Gibco BRL). The cells were passaged every 3 days. For the evaluation of NO synthesis, the cells were suspended in culture medium to give 4 x 10’ cells/ml and placed in 48-well plates at 0.5 mL. For evaluation of HO-1 induction, the cells were suspended in culture medium to give 3 x lo5 cells/ml and placed in 6-well plates at 5 mL. After overnight culture, fresh medium was added, and the assayswere conducted. Evaluation of HO-1 induction To the RAW264.7 cells, various concentrations of Agaro-oligosaccharide solutions were added and cultured for 12 hr. At the end of the culture period, the cells were collected, washed in PBS, resuspended in lysis buffer (0.1% Triton X-100, 10 mM EDTA-2Na, 1 mM PMSF, 0.2 mM leupeptin, 0.05 mM pepstatin A in 100 mM Tris HCl, pH 7.4), and subjected to one cycle of freeze-thaw to prepare the cell extract. This was then centrifuged at 4”C, 10,000 ‘ pm, for 20 min, and the supematant was collected. The protein concentration in the samples was measured using the Micro BCA protein assay reagent (Pierce Chemical Company). 12.5% polyacrylamide gel. Cell extract containing 10 ,ug of the protein was mixed with the Laemmli loading buffer, heated at 100°C for 5 min, and applied to PVDF membrane (Millipore After electrophoresis, the proteins were transferred to Japan). The signal was detected by chemiluminescence
(Renaissance, NEN Life Science Products) using anti-HO-l polyclonal antibody (Santa Cruz Biotechnology), with anti- o tubulin monoclonal antibody (Calbiochem) as the
internal control. Evaluation for 5 hr. of NO synthesis LPS was then added to a final concentration of 1 ,ug/mL and IFN- a! to final After the incubation period, the
Agarobiose was added to RAW264.7 cells at various concentrations and cultured concentration of 10 U/mL and cultured for 16 hr. of NO synthesized.
culture medium was assayed for nitrite formed as a result of NO hydrolysis as an index 100 PL of the culture supernatant was treated with 100 PL of the diamine in 5% 4% Griess reagent (1% sulfanilamide, 0.1% N-(1-naphthyl-)ethylene
o-phosphoric acid, Sigma) and measured after 15 min in a plate reader at 540 nm absorbance. Isolation and storage of human peripheral blood mononuclear cells (PBMC) 400 mL of blood was obtained from human healthy donors. The blood was diluted 2 fold in PBS(-), overlaid on Ficoll-paque (Pharmacia) and centrifuged for 20 min at 500 x g. The peripheral blood mononuclear cells (PBMC) at the interphase were collected with a pipette and washed in RPM11640 medium (BioWhittaker). The PBMC collected was resuspended in the freezing solution of 90% FCS (JRH Biosciences)/lO% dimethyl sulfoxide, and stored in liquid nitrogen. At the time of the experiment, the PBMC stored was rapidly thawed in a 37°C water bath, washed in RPM11640 medium containing 10 pg/mL DNase (Calbiochem), and cell viability tested...
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