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yyc12596新虫 (著名写手)
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小鼠腹腔中性粒细胞 已有1人参与
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小鼠腹腔中性粒细胞。 没有太多实际参考，找文献里的方法看看。 |
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yyc12596
新虫 (著名写手)
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文献1： Prostaglandin E2 Receptors, EP2 and EP4, Differentially Modulate TNF-α and IL-6 Production Induced by Lipopolysaccharide in Mouse Peritoneal Neutrophils ☆ Hana Yamane, Yukihiko Sugimoto, Satoshi Tanaka, Atsushi Ichikawa1 BBRC,Volume 278, Issue 1, 11 November 2000, Pages 224–228 关于小鼠腹腔中性粒细胞的获得方法： Preparation of peritoneal neutrophils. Mice were injected intraperitoneally with 2 ml of 5% casein in sterile saline and were killed by cervical dislocation 5–6 h after injection. The lavage fluids were collected in a syringe, and exudated peritoneal cells were precipitated by centrifugation. Neutrophils in peritoneal cells were purified by Percoll stepwise density gradient (1.090 and 1.070 g/ml) centrifugation (600g for 20 min at 4°C). The purity of neutrophils was greater than 95% as determined by staining with May–Grunwald– Giemsa. Neutrophils were suspended in RPMI 1640 medium containing 10% heat-inactivated fetal bovine serum, 150 mM 2-mercaptoethanol and 100 mM sodium pyruvate. 关于因子刺激： Measurement of cytokine production. Neutrophils (1 3 106 cells/ ml) were incubated with or without 100 ng/ml LPS for the indicated time at 37°C in 5% CO2. After incubation, each culture was centrifuged at 300g for 5 min at 4°C to remove the cells. The amounts of TNF-a and IL-6 in the supernatant were assayed using the respective ELISA kits according to the manufacturer’s instructions. |
2楼2014-10-03 23:43:36
yyc12596
新虫 (著名写手)
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结果： Effect of PGE2 on LPS-Stimulated TNF-a and IL-6 Production in Mouse Peritoneal Neutrophils LPS treatment (100 ng/ml) induced the production of TNF-a and IL-6 in mouse peritoneal neutrophils (Fig. 3). TNF-a production rapidly increased and then decreased to the basal level by 12 h. The maximum level was obtained at 2 h after LPS stimulation. However, IL-6 production increased gradually and reached a plateau level at 8 h after LPS stimulation. Simultaneous addition of PGE2 with LPS suppressed the TNF-a production and enhanced the IL-6 production significantly (Fig. 3). However, the PGE2 effects in the absence of LPS were relatively small. Dibutyryl cAMP (1 mM) suppressed the TNF-a production at 2 h and enhanced the IL-6 production at 4 hr after the LPS stimulation in peritoneal neutrophils (Table I). It was reported that endogenous PG synthesis was induced by LPS treatment through COX-2 induction in human neutrophils [16, 17]. However, in mouse peritoneal neutrophils pretreatment with indomethacin (1 mM), a nonselective COX inhibitor, induced no changes in the LPS-stimulated production of TNF-a and IL-6 (data not shown). 这篇文章比较简单。再搜搜其他文献看。 |
3楼2014-10-03 23:47:08
yyc12596
新虫 (著名写手)
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文献2： Enhanced Th1 Activity and Development of Chronic Enterocolitis in Mice Devoid of Stat3 in Macrophages and Neutrophils Kiyoshi Takeda1, 4, 2, Björn E Clausen2, 3, 3, Tsuneyasu Kaisho1, 4, Tohru Tsujimura2, Nobuyuki Terada2, Irmgard Förster3, 2, 4, Shizuo Akira1, 4, 2, 1 Immunity的一篇文章。这家杂志影响因子过20分了，几乎是BBRC的十倍。 Volume 10, Issue 1, 1 January 1999, Pages 39–49 获取中性粒细胞的方法： For peritoneal neutrophils, cells were isolated from the peritoneal cavity after 4 hr of thioglycollate injection and cultured for 18 hr in the presence or absence of 10 ng/ml IL-10. Then, nonadherent cells were harvested and used for further experiments. These cells were >90% positive for Gr-1 as determined by flow cytometry. In the case of more strict experiments, the cells were further enriched by magnetic cell sorting (MACS, Miltenyi Biotec) using biotin-conjugated anti-Gr-1 antibody and streptavidin-microbeads. |
4楼2014-10-03 23:55:15
yyc12596
新虫 (著名写手)
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刺激实验： (E) Peritoneal cells from mice injected with thioglycollate 4 hr prior to cell harvest were cultured with or without 10 ng/ml IL-10 for 12 hr. Nonadherent neutrophils were collected and stimulated with PMA for 1 hr or with LPS for 24 hr. After PMA stimulation, H2O2 production was determined based on reduction of ferricytochrome C. After LPS stimulation, TNFα production was measured by ELISA. |
5楼2014-10-04 00:04:17
yyc12596
新虫 (著名写手)
- 应助: 10 (幼儿园)
- 金币: 10847.3
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- 虫号: 2034994
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6楼2014-10-04 00:26:50
yyc12596
新虫 (著名写手)
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文献3： Caspase 1–Independent Activation of Interleukin-1 in Neutrophil-Predominant Inflammation Monica Guma,1 Lisa Ronacher,1 Ru Liu-Bryan,2 Shinji Takai,3 Michael Karin,1 and Maripat Corr1 ARTHRITIS & RHEUMATISM Vol. 60, No. 12, December 2009, pp 3642–3650 DOI 10.1002/art.24959 中性粒细胞的获取方法： Neutrophil isolation. Mice were injected IP with 1 ml of 3% thioglycolate (Difco, Franklin Lakes, NJ). After 3–5 hours, the mice were killed, and peritoneal cells were removed by lavage with 5 ml of 3 mM EDTA in PBS. The cells were incubated with anti-CD16/CD32 (BD Biosciences) and then with phycoerythrin (PE)–labeled anti–Gr-1 (BD Biosciences). The cells were magnetically separated using anti-PE–coated beads (Miltenyi Biotec, Auburn, CA) in accordance with the manufacturer’s instructions. Cell purity was verified to be 95% by flow cytometry. The cells were seeded into 96-well plates at a density of 250,000/well. |
7楼2014-10-04 00:38:19
yyc12596
新虫 (著名写手)
- 应助: 10 (幼儿园)
- 金币: 10847.3
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8楼2014-10-04 00:39:40
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