3.1 Chemicals
The powdered fruiting bodies of A. cinnamomea (POZO-4, BCRC930103, DNA sequencing listed in NCBI data bank, 18S ribosomal gene sequencing recorded number EU077558~61 and ITS ribosomal gene recorded number EU077562~65) are purchased from Po-Zone Enterprises Co., LTD (New Taipei City, Taiwan). The powdered fruiting bodies of A. cinnamomea (100 g) were boiled in the 1000 mL of distilled water for 3 hr. The water extract of fruiting bodies of A. cinnamomea (ACW)
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filtered with 0.45 µm was analyzed by high-performance liquid chromatography (HPLC, Hewlett Packard 1100 series). The HPLC fingerprint of ACW was performed by Medical and Pharmaceutical Industry Technology and Development Center (Taipei, Taiwan). In the animal experiment, the ACW was given orally to rats at a dose of 6.67 mg/kg body weight/day for 8 weeks as described previously [23]. Control rats were given access to water alone. The reagents of H2O2, and N-nitrosodiethylamine (DEN) were purchased from Sigma Chemical Co. (St. Louis, MO, USA). AC (Figure 2) was isolated from the fruiting bodies of A. cinnamomea as described previously [24] and provided by Po-Zone Enterprises Co., LTD (New Taipei City, Taiwan).
3.2 Experimental model of liver injury
Total 108 male Wistar rats (200-250 g) were housed at the Experimental Animal Center, National Taiwan Normal University, at a constant temperature and with a consistent light cycle (light from 07:00 to 18:00 O'clock). Food and water were provided ad libitum. All surgical and experimental procedures were approved by National Taiwan Normal University Animal Care and Use Committee and were in accordance with the guidelines of the National Science Council of Republic of China (NSC 1997). The grouping and experimental design were indicated in Figure 1. To evaluate the co-treating effect of ACW or AC on DEN-induced liver injury, 72 animals were randomly divided into ten groups, group 1: control group (Con, n=18);
group 2 with 2 weeks of DEN treatment (2WDEN, n=6); group 3 with 4 weeks of DEN treatment (4WDEN, n=6); group 4 with 8 weeks of DEN treatment (8WDEN, n=6); group 5 with 2 weeks of ACW and DEN treatment (2WDEN+ACW, n=6);
group 6 with 4 weeks of ACW and DEN treatment (4WDEN+ACW, n=6), group 7 with 8 weeks of ACW and DEN treatment (8WDEN+ACW, n=6), group 8 with 2WDEN treatment plus antcin K (AC, 20 µg/kg body weight) co-treatment
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(2WDEN+AC, n=6), group 9 with 4WDEN treatment plus AC co-treatment (4WDEN+AC, n=6) and group 10 with 8WDEN treatment plus AC co-treatment (8WDEN+AC, n=6).
For exploration of the therapeutic potential of ACW, we also included the effect of post-treatment of ACW after 2 weeks of DEN injury in the rats subjected to 2 weeks (2WDEN+PACW, n=6), 4 weeks (4WDEN+PACW, n=6) and 8 weeks of DEN injury (8WDEN+PACW, n=6). For reducing the animal number in this study, the control group was used the group 1 as described above. In another 18 rats, an effective silymarin treatment (200 mg/kg) on DEN-treated hepatic injury was performed in the 2WDEN+silymarin (n=6), 4WDEN+silymarin (n=6) and 8WDEN+silymarin (n=6) groups. DEN was given as an initiator of liver injury at 500 ppm in the drinking water throughout the entire experiment (8 weeks). ACW at a dosage of 6.67 mg/kg body weight was given by daily gavage twice for 8 weeks.
3.3 In vivo and in vitro chemiluminescence recording for ROS activity
The in vivo ROS generation in response to DEN injury was measured from the liver surface by intravenous infusion of a superoxide anion probe, 2-Methyl-6-(4-methoxyphenyl)-3,7-dihydroimidazo- [1,2-a]-pyrazin- 3-one-hydrochloride (MCLA) (0.2 mg/ml/h, TCI-Ace, Tokyo Kasei Kogyo Co. Ltd., Tokyo, Japan) and by the use of a Chemiluminescence Analyzing System (CLD-110, Tohoku Electronic In. Co., Sendai, Japan) [9]. MCLA, lucigenin and luminol are very sensitive chemiluminescence probes used to detect ROS and reactive nitrogen species formation. Briefly, the rat was maintained on a respirator and a circulating water pad at 37°C in a dark box with a shielded plate. Only the liver window was left unshielded and was positioned under a reflector, which reflects the photons from the exposed liver surface by the MCLA-amplified chemiluminescence onto the detector area. The
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real-time displayed chemiluminescence signal was indicated as O2-. ROS level from the liver surface.
The measurement of bile ROS or in vitro experiment was also detected by a lucigenin-amplification for O2-. determination [9] and luminol-amplification for H2O2
measurement [34]. In brief, the bile samples were immediately wrapped in aluminum foil and kept on ice until chemiluminescence measurement, usually done within 2 h [54]. Immediately before ROS measurement, 0.1 mL of phosphate-buffered saline (pH 7.4) was added to 0.2 ml of bile sample. The chemiluminescence was measured in a completely dark chamber of the Chemiluminescence Analyzing System (CLD-110). After 100-s background level determination, 1.0 mL of 0.1 mM lucigenin [54] or 1.0 mL of 0.2 mM luminol in phosphate-buffered saline (pH 7.4) was injected into the sample. The ROS signal was monitored continuously for an additional 300 s.
The total amount of O2-. or H2O2 chemiluminescence was calculated by integrating of the area under the curve and subtracting it from the background level.
Three major ROS like O2-., hydrogen peroxide (H2O2) and nitric oxide (NO) can initiate inflammation [9,35]. Therefore, we determined these three ROS in cell-free system in vitro, respectively. Superoxide generation was measured in a cell-free enzyme system by lucigenin-enhanced chemiluminescence. Assay solutions consisting of lucigenin (0.1 mM in 1.0 mL) and xanthine (10−5 M in 100 µl) were prepared in Krebs-HEPES buffer (composition (mM): NaCl 99.0, KCl 4.7, KH2PO4
1.0, MgSO4·7H2O 1.2, D-glucose 11.0, NaHCO3 25.0, CaCl2·2H2O 2.5, Na-HEPES 20.0) [36]. Aliquots (0.2 mL) of the assay solutions containing distilled water, different dosage of ACW or AC were placed into the CLD-110 chemiluminescence analyzer to measure background photon emission over 30 sec. After background counting was completed, xanthine oxidase (5 × 10−6 M, final concentration 10 U/mL)
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was added to the mixture and photon emission, as a measure of O2− production, was counted for 300 sec.
For H2O2 determination, chemiluminescent signals emitted from the test mixture containing PBS, different dosage of ACW or AC and 50 µL of H2O2 (0.03 %) were amplified by 1.0 mL of 0.2 mM luminol (Sigma) was measured with a ultrasensitive chemiluminescent analyzer (CLD-110) and the same protocol as O2-. detection [34].
For NO measurement, the NO chemiluminescence counts were examined in the test mixture containing the chemiluminescent probe 0.1 mM luminol, 10 mM H2O2, desferrioxamine 0.15 mM and K2CO3 2 mM. A NO probe in sodium phosphate buffer (0.1 M; pH 7.4) was prepared from zwitterionic polyamine/NO adducts for releasing NO including 1-hydroxy-2-oxo-3-(3-aminopropyl)-3-isopropyl-l-triazene (NOC5) and 1-hydroxy-2-oxo-3-(N-methyl-3-aminopropyl)-3-methyl- 1-triazene (NOC7) (Dojin Chemicals Co., Ltd., Kumamoto, Japan) [37]. The different dosage of ACW or AC was administered in the NO probe. After measuring the background of chemiluminescent solution mixture, the NO probe was added into the chemiluminescent solution and the chemiluminescent counts were detected by a chemiluminescence analyzer (CLD-110). All the assays were performed in six times for each sample, and total chemiluminescence counts in 300 s were calculated by integrating the area under the curve.
3.4 Inflammation, ED-1, 3-NT, 4-HNE, apoptosis and autophagy in the liver
The histologic features were assessed and scored for inflammation (portal and lobular) by hematoxylin and eosin and fibrosis by Masson trichrome as following.
The grade of lobular inflammation was scored as 0 = no foci, 1 = < 2 foci/200xfield, 2
= 2-4 foci/200xfield, and 3 = > 4 foci/200xfield. Fibrosis was scored as 0 = none, 1 = zone 3 perisinusoidal fibrosis or portal/periportal, 2 = perisinusoidal and
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portal/periportal fibrosis, 3 = bridging fibrosis, and 4 = cirrhosis [25].
We suggested that the high levels of ROS might promote hepatic accumulation of leukocytes, nitrated protein and lipid peroxides. We immunostained 3-nityrotyrosine (3-NT), 4-hydroxynonenal (4-HNE), and ED-1 in the paraffin-embedded sections of liver tissues [9]. For these oxidative injury measurement, the rats (n=6 in each group) were sacrificed at the end of experiment.
Hepatic sections were deparaffinized, rehydrated, and stained immunohistochemically for presence of in vivo markers of lipid peroxidation, 4-HNE-protein adducts, by incubation with a polyclonal antibody (Alpha Diagnostic International; San Antonio, TX, USA) and with rabbit polyclonal anti-3-NT antibody (Alpha Diagnostic International; San Antonio, TX, USA) diluted at 1:50. The percentage of 3-NT, or 4-HNE expression was calculated as 3-NT-, or 4-HNE-stained area/total area × 100%
and analyzed by Adobe Photoshop 7.0.1 image software analysis.
For hepatic macrophage (ED-1) staining, the tissue sections were incubated overnight at 4°C with a mouse anti-rat antibody to ED-1 (CD68, 1:200, Serotec, Sydney, NSW, Australia). A biotinylated secondary antibody (Dako, Botany, NSW, Australia) was then applied followed by streptavidin conjugated to HRP (Dako). The chromogen used was Dako Liquid diaminobenzene (DAB). Twenty high-power (×400) fields were randomly selected for each liver section, and the value of ED-1 positive cells was counted.
We performed Beclin-1-related autophagy and terminal deoxynucleotidyl transferase-mediated nick-end labeling (TUNEL) apoptosis method [10] to investigate the presence and extent of two types of programmed cell death in DEN-induced hepatic injury. The hepatic sections (5 µm) were prepared, deparaffinized, and stained by the hematoxylin & eosin, Beclin-1staing, and TUNEL-avidin-biotin-complex
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methods. A biotinylated secondary antibody (Dako, Botany, NSW, Australia) was then applied followed by streptavidin conjugated to HRP (Dako). The chromogen used was Dako Liquid diaminobenzene (DAB). Twenty high-power (×400) fields were randomly selected for each section, and the value of each oxidative stress was analyzed using a Sonix Image Setup (Sonix Technology Co., Ltd) containing image analyzing software Carl Zeiss AxioVision Rel.4.8.2 (Future Optics & Tech. Co. Ltd., Hangzhou, China).
3.5 Western Blotting for p85, MAPK and CYP2E1 in the livers
The liver protein concentration was determined by a BioRad Protein Assay (BioRad Laboratories, Hercules, CA, USA). Ten µg of protein was electrophoresed as described previously [9]. The expression of phosphorylated p85, phosphorylated MAPK, and CYP2E1 in liver tissue was evaluated by Western immunoblotting and densitometry. Briefly, the total proteins were homogenized with a prechilled mortar and pestle in extraction buffer, which consisted of 10 mM Tris-HCl (pH 7.6), 140 mM NaCl, 1 mM phenylmethyl sulfonyl fluoride, 1% Nonidet P-40, 0.5% deoxycholate, 2% β-mercaptoethanol, 10 µg/ml pepstatin A, and 10 µg/ml aprotinin. The mixtures were homogenized completely by vortexing and kept at 4°C for 30 min. The homogenate was centrifuged at 12,000 g for 12 min at 4°C, the supernatant was collected, and the protein concentrations were determined by BioRad Protein Assay (BioRad Laboratories). Antibodies raised against cytochrome P450 CYP2E1 (Chemicon International Inc., Temecular, CA, USA), phosphorylated p85 (Anti-PI3K p85, phosphor Y607, Abcam, Cambridge, MS, USA), phospho-p44/42 MAPK (Erk1/2, Thr202/Tyr204, Cell Signaling, Danver, MA, USA) and monoclonal mouse antimouse β-actin (Sigma, Saint Louis, MI, USA) were used at 1:400. All of these antibodies cross-react with the respective rat antigens. Proteins on SDS-PAGE gels
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were transferred to nitrocellulose filters and stained as described. The density of the band with the appropriate molecular mass was determined semi-quantitatively by densitometry using an image analyzing system (Alpha Innotech, San Leandro, CA).
3.6 Preparation of Monascus adlay (MA)
Monascus fermented products were prepared on an adlay substrate, using a solid-state culture method as described previously [41,48]. In brief, adlay (obtained from the Erhlin Farmers’ Association, Changhua County, Taiwan) was immersed in deionized water for 2 h, dried and autoclaved at 121°C for 20 min. We inoculated M.
purpureus Went (CCRC 31498; obtained from the Culture Collection and Research Center, Food Industry Research and Development Institute, Hsinchu City, Taiwan) onto malt extract agar (Difco agar, Voigt Global Distribution Inc, Kansas City, Missouri, USA) at 25°C for 72 h. We inoculated the mycelium into potato dextrose broth (Difco) and incubated it at 25°C for 7 d. The culture was homogenized in a blender and inoculated into autoclaved adlay with 10% M. purpureus Went at an inoculation rate of 5%. MA products were harvested after the fungal mycelia had colonized for 7 d at 25°C. MA was autoclaved at 121°C for 20 min and air-dried in an oven at 40°C. The dried MA was ground into a coarse powder (20 mesh) using a Restsch Ultra Centrifugal Mill and Sieving Machine (Haan, Germany). Our previous report had indicated the MK content in MA extract by a high-performance liquid chromatography (HPLC) [48]. Briefly, 1 g of powdered MA was extracted with 5 mL ethyl acetate at 70°C for 1.5 h. After 5 min of centrifugation at 1800 rpm and filtration through a 0.45-µm membrane, the filtrate was dried under a vacuum. One milliliter of acetonitrile was added to the resulting mixture, which was then filtered with a 0.45-µm pore size filter and analyzed by an HPLC system (Model L-6200, Hitachi, Japan). MK content in our prepared MA extract was 1.57 mg/g (0.157%).
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3.7 Animals and MA Treatments
The onset of hemolysis and subsequent thrombosis and tissue infarction is faster in female than in male rats [53]. Therefore, we explored the medical efficacy of an MA diet and MK on 24 female Wistar rats (200–220 g; mean body weight, 206 ± 10 g) housed at the Experimental Animal Center of National Taiwan Normal University.
The standard rat chow diet contained 58% carbohydrates, 28.5% proteins, and 13.5%
fat (Laboratory Rodent diet 5001; Young Li Trading Company Ltd., Sijhih City, New Taipei City, Taiwan). We mixed powdered rat chow diet and powdered MA at a ratio of 99:1 by combining them through a 20-mesh sieve (aperture = 0.94 mm) [48]. We used 2% corn starch and 3% soybean oil to re-form the product into the MA lump diet.
The same methods were used to crush and re-form the control, standard rat chow diet into food lumps. Animals were provided with food and tap water ad libitum.
3.8 FeCl3-induced carotid arterial time to occlusion (TTO)
All the rats were anesthetized by subcutaneous injectionof 1.2 g/kg urethane (Sigma-Aldrich Inc., St. Louis, MO, USA). After arterial isolation, transonic flow probes (Probe# 0.5VBB517, Transonic Systems Inc., Ithaca, NY, USA) for carotid arterial blood flow measurement were applied and displayed on a small animal blood flow meter (Model 206, Transonic Systems Inc., Ithaca, NY, USA). All the blood flow signals were continuously recorded with an ADI System (PowerLab/16S, ADI Instruments, Pty Ltd, Castle Hill, Australia). The carotid arteries were injured as previouslydescribed11 with a slight modification. In brief, a filter paper (1 mm ×2 mm), soaked with 30% FeCl3 solution (Ferric chloride, Sigma, St. Louis, MO, USA), was applied to the artery for 3 minutes and the cavity was filled with saline immediately. The flow rate was continuously recorded, and the time to occlusion (TTO, arterial blood flow decreases to zero) was determined. For some animals, the arterial rings were fixed in 10% formalin after completing the thrombosis protocol, as
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described previously [48]. The injured arterial segments were excised, embedded in paraffin, sectioned, and subjected to hematoxylin and eosin staining.
3.9 Grouping
Rats were randomly divided into the following groups: control diet without TTO (n=6), control diet with TTO (n=6), MA diet with TTO (n=6), and MK pretreatment with TTO (n=6). Both MA and MK treatments lasted for 2 weeks. MK (lovastatin) (A.G. Scientific, CA, USA) was dissolved in deionized water at a concentration of 1.0 mg/100 mL. We found that rats in the MA group ingested 30±3 g apiece, or approximately 0.47 mg of MK per rat [48]. Animals in the MK group drank approximately 40-50 mL of treated water apiece, for a mean dosage of MK around 0.47 mg/rat. These doses were based on Boyd's formula for body surface area, such that daily doses of MA and MK were equivalent to the daily recommended supplemental doses for adult humans (~2 g of Monascus-fermented products and ~20 mg of MK).
All surgical and experimental procedures were approved by the Institutional Animal Care and Use Committee of National Taiwan Normal University and are in accordance with the guidelines of the National Science Council of the Republic of China (NSC 1997). All possible efforts were made to reduce the numbers of animals used and to minimize animal suffering during the experiment.
3.10 Lucigenin-Enhanced Chemiluminescence (CL) Counts
We considered that MA or MK may affect the degree and process of oxidative stress in the carotid arteries. We selected 900 sec of FeCl3 lesion to the carotid artery to determine the degree of ROS amount before its complete occlusion. We used lucigenin- and luminol-amplified chemiluminescence (CL) methods detect O2-., H2O2
and HOCl amounts in MA (65 mg/mL containing 0.1 mg MK), and MK (0.1 mg/mL).
These data were compared to the control values measured in distilled water [54]. The
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lucigenin-enhanced CL method has been confirmed to be a reliable assay for oxidative stress in damaged tissue [54]. We compared the differences in CL counts from carotid arteries in the rat subjected FeCl3 injury as well as the MA or MK treatment. The carotid artery was removed after each treatment and homogenized with saline in a 0.1 mL volume. ROS levels in the homogenized arteries were determined by a CL analyzer (CLD-110; Tohoku Electronic Industrial, Japan) after administration of 1.0 ml of 0.1 mM lucigenin in phosphate-buffered saline (pH 7.4) into the tested samples. The assay was performed in duplicate for each sample, and total CL counts in 600 s were calculated by integrating the area under the curve.
3.11 Soluble form of Vascular ICAM-1 and ROS assay
To obtain the quantified data of oxidative stress, in some rats after FeCl3 lesion for 900 s, the homogenates of arterial rings were used for measurement of soluble form of ICAM-1 (sICAM-1) by an ELISA kit (rat ICAM-1/CD54 Quantikine ELISA Kit) and H2O2–ROS amount by a luminol-amplified CL assay as described above.
sICAM-1 is an important biomarker and the main cause for neutrophil adhesion to endothelium then to release ROS and to trigger thrombosis in the vessel wall [48,51].
3.12 In situ demonstration of ROS production and amount in the carotid artery
High levels of ROS might promote the expression of 3-nitrotyrosine (3-NT) and ICAM-1 in the endothelium to trigger thrombotic cascades. We considered that MA or MK may affect the degree and process of oxidative stress in the carotid arteries. We selected 900 s of FeCl3 lesion to the carotid artery to determine the degree of oxidative stress 3-NT and ICAM-1 by immunocytochemical stains. For immunocytochemical stains, the rats (n=3 in each group) were sacrificed at the end of experiment. 3-NT and the ICAM-1 [55] expression in the paraffin-embedded sections of the vascular rings were immunostained. The 5-µm cross-sections were stained with
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anti-3-NT antibody (Alpha Diagnostic International; San Antonio, TX) and with ICAM-1 antibody (R&D Systems, Minneapolis, MN). The 3-NT and ICAM-1 stains were photographed on a Leica microscope (Leica Microsystems Wetzlar, Wetzlar, Germany).
The percentage of staining in the vascular rings was calculated by the formula:
% staining = stained curved length/total curved length.
The thrombus size was determined by Adobe Photoshop 7.0.1 imaging software using the following formula:
% thrombus size = thrombus area of intravascular area/total intravascular area × 100
3.13 Immunoblot analysis for NFκB, 3-NT, ICAM-1, CHOP, and Nrf2
To explore proteins expression, we selected 4 h of FeCl3 lesion to the carotid artery to determine the degree of proinflammation transcription factor, NF-κB, oxidative stress biomarkers, 3-NT and ICAM-1, endoplasmic reticulum stress biomarker, CCAAT/-enhancer-binding protein homologous protein (CHOP), by western blotting. The immunoblotting method for western blotting was performed as described previously [51]. We determined the expression of 3-NT, ICAM-1, CHOP and β-actin in the total homogenates of carotid arterial tissues subjected to 30% FeCl3
lesion. We evaluated NF-κB p65 and Nrf2 in the nuclear proteins. The isolated arteries were placed in ice-cold isolation buffer containing 0.5 M sacarose, 10 mM Tris-HCl, 1.5 mM MgCl2, 10 mM KCl, 10% glycerol, 1 mM EDTA, 1 mM DTT, 2 mg/mL aprotinin, 4 mg/mL leupeptin, 2 mg/mL chymostatin, 2 mg/mL pepstatin, and 100 mg/mL 4-(2 aminoethyl)- benzenesulfonyl fluoride at pH 7.4 and were homogenized by using a tissue grinder. Then, the supernatant was resuspended in isolation buffer and the aliquots (nuclear fractions) were stored at -70°C. Antibodies
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raised against NFκB (R&D Systems, Minneapolis, MN), LaminA/C, Nrf2, CHOP (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), 3-NT (Alpha Diagnostic International, San Antonio, TX), ICAM-1 (R&D Systems, Minneapolis, MN, USA) and β-actin(catalog no. A5316, clone AC-74, Sigma) were used. The immunoreactive bands were detected by incubation with each respective antibody; the secondary antibody alkaline phosphatase; and, finally, nitroblue tetrazolium, 5-bromo-4-chloro-3-indolyl phosphate, and a toluidine salt (Roche Diagnostic, Mannheim, Germany)stock solution for 30 minutes at roomtemperature.
3.14 Preparation of Platelet Suspensions for Platelet activation and aggregation
Rat platelet suspensions were prepared as described previously [56]. In brief, blood (5 mL) was drawn from the carotid artery into plastic tubes containing 1 mL of 3.8% sodium citrate buffer (blood : sodium citrate = 9 : 1). Platelet-rich plasma was obtained by low-speed centrifugation (1500 rpm for 10 min) and further centrifuged at 15,000 rpm for 10 min to obtain a platelet pellet. The platelets were suspended in of 190 µL aliquots of Ca2+-free Tyrode's solution (pH 7.35) and were incubated with the indicated concentrations of MA extract, MK or vehicle (DMSO) for 30 min at 37 °C.
MA or MK was dissolved in DMSO as a stock solution and stored at − 20 °C. The platelet number was adjusted to 3 × 108/mL before use. A turbidimetric method was adapted to measure platelet aggregation with a lumi-aggregometer (Payton Scientific, Scarborough, ON, Canada). Specifically, 10 µL of 2 mM ADP was administered to the platelet suspension (0.4 mL) for 5 min and the response of aggregation was expressed in light-transmission units.
For platelet activation, the washed platelets, pretreated with MA or MK were stimulated with collagen (2.5 µg/mL) and incubated for 5 min at 37°C. A detailed procedure was described below. The reaction was terminated, and the platelets were
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centrifuged followed by resuspension in ice-cold phosphate buffere saline (PBS) containing 10% fetal calf serum (FCS) and 1% sodium azide. The platelets were incubated with CD62P primary antibody (P-selectin, Thermo Fisher Scientific Taiwan, Co., Ltd., Taipei, Taiwan) in 3% bovine serum albumin (BSA)/PBS for 30 min at 4
°C in the dark, then fixed and washed three times by centrifugation at 400×g for 5 min.
The platelets were resuspended in ice-cold PBS followed by FITC-conjugated secondary antibody (Santa Cruz Biotechnology) incubation in 3% BSA/PBS for 30 min at 4 °C in the dark. Platelets were again washed three times by centrifugation at 400×g for 5 min and resuspended in ice-cold PBS, 3% BSA and 1% sodium azide.
The samples were analyzed with a FACSCalibur flow cytometer using CellQuest
The samples were analyzed with a FACSCalibur flow cytometer using CellQuest