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小木虫论坛-学术科研互动平台 » 生物医药区 » 生物科学 » 细胞科学 » 小鼠腹腔中性粒细胞
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yyc12596

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[交流] 小鼠腹腔中性粒细胞 已有1人参与

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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1楼 2014-10-03 23:40:33
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yyc12596

新虫 (著名写手)
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.
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2楼2014-10-03 23:43:36
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yyc12596

新虫 (著名写手)
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。
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3楼2014-10-03 23:47:08
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yyc12596

新虫 (著名写手)
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.
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4楼2014-10-03 23:55:15
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yyc12596

新虫 (著名写手)
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.
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5楼2014-10-04 00:04:17
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yyc12596

新虫 (著名写手)
搜文献的能力又被鄙视了。
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6楼2014-10-04 00:26:50
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yyc12596

新虫 (著名写手)
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.
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7楼2014-10-04 00:38:19
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yyc12596

新虫 (著名写手)
刺激实验：

C,Thioglycolate-induced neutrophils from C57BL/6 WT mice (n  3)
were stimulated with or without LPS (100 ng/ml) in the presence or
absence of elastase inhibitor III (MeOSuc-AAPV-cmk) (500 M).
After 6 hours and 16 hours, the levels of IL-1 secretion were
measured by ELISA.
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8楼2014-10-04 00:39:40
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lfh123lfh321

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很有帮助，感谢
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9楼2020-04-25 11:12:34
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