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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 » 综合其他 » microvesicle ultracentrafuge
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yyc12596

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[交流] microvesicle ultracentrafuge 已有1人参与

microvesicle获取。
   文献中有用AnnexinV磁珠拉下来的。
    但是据说ultracentrafuge也可以。对于超声离心不熟悉，查查资料看看吧。
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1楼 2014-08-04 23:24:56
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yyc12596

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★ ★ ★
youlinglyw: 金币+3, 赠人玫瑰手有余香，生物科学有你更精彩！ 2014-08-05 09:42:24
实验室的Ultracentrifuge是Beckman Coulter的Optima™ L-80 XP。
我之前没用过。

公司对于仪器的说明网址：
https://www.beckmancoulter.com/w ... /2/392049///0/1//0/
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2楼2014-08-04 23:31:26
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yyc12596

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配套的软件：
Optima's easy-to-use eXPert Software gives your lab an edge by making it possible to quickly design and optimize separation protocols using a combination of rotors and tubes to simulate results — all before performing separations. A unique slide-bar feature allows you to view the separation at earlier run times to better suit the isolation of individual components. Up to three components can be modeled using the rate-zonal protocol. Then save the simulated run and download it with a single click of the mouse when you're ready to begin your run. Optima eXPert Software's advanced intelligence delivers greater productivity and faster turnarounds for your lab.

The following demonstrations illustrate just how easy it is to optimize efficiency with Optima eXPert Software.
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3楼2014-08-04 23:34:31
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yyc12596

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设置时间：
Optima eXPert Software Demo 2:
Run Simulation -- Rate Zonal

Find out how to stop your run at the optimum separation time before the sample material reaches the tube bottom and becomes a pellet.
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4楼2014-08-04 23:35:51
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yyc12596

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文献1的离心方法：

Ultracentrifugation and microvesicle isolation

Microvesicle-free supernatant was generated by ultracentrifugation
(2.5 h, 100,000 g) and subsequently used for ELISA
analysis. To isolate microvesicles, cell supernatant from senescent
cells was subjected to serial ultracentrifugation. Briefly,
conditioned, serum-free cell supernatant was first cleared
from dead cells and debris by low-speed centrifugation (10
min., 1000 g) and afterward spun for 30 min at 10,000 g to
precipitate larger vesicles. In order to enrich for exosomes,
the resulting supernatant was filtered through a 0.2 m filter
prior to ultracentrifugation at 100,000 g for 2.5 h (Optima
LE-80K, Type 70 Ti Rotor; Beckman Coulter, Krefeld, Germany).
The microvesicle pellet was washed with PBS, transferred
to cell lysis buffer, and used for Western blotting.
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5楼2014-08-04 23:43:28
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yyc12596

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Senescence-associated release of transmembrane proteins involves proteolytic processing by ADAM17
and microvesicle shedding
Timo Effenberger,*,1 Jan von der Heyde,*,1 Kareen Bartsch,* Christoph Garbers,*
Klaus Schulze-Osthoff,†,‡,§ Athena Chalaris,* Gillian Murphy, Stefan Rose-John,*
and Björn Rabe*,2
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6楼2014-08-04 23:44:47
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yyc12596

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尿样里用超声离心提取microvesicle的protocol：

Methods in Molecular Biology Volume 1024, 2013, pp 211-220 Date: 05 May 2013

Methods of MicroRNA Quantification in Urinary Sediment

Gang Wang, Cheuk Chun Szeto
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7楼2014-08-05 00:03:59
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yyc12596

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尿样准备：
1. Collect fresh urine in a 500 ml sterile plastic bottle ( see Note 2 ).
2. Transfer fresh urine to 50 ml polypropylene centrifuge tubes( see Note 3 ).
3. Centrifuge for 30 min at 3,000 × g , at 4 °C, using refrigerated centrifuge with swinging-bucket rotors.
4. Pipet off the supernatant, and dispense into 2-ml screw-cap Eppendorf ultracentrifuge tubes. Discard sediment and dispose the 50-ml polypropylene centrifuge tubes properly ( see Note 4 ).
5. Mark the cap of each ultracentrifuge tube with a waterproof mark.
6. Put the tubes in the fi xed-angle rotor of tabletop ultracentrifuge.
7. Orient the tubes in the rotor and make the waterproof mark face up ( see Note 5 ).
8. Close tabletop ultracentrifuge and centrifuge for 30 min at 13,000 × g , at 4 °C.
9. Take out the tube, note the position of cell debris, and carefully draw the supernatant with 1 ml syringe without disturbing the sediment ( see Note 6 ).
10. Inject the supernatant into 0.22 μ m fi lter and collect the supernatant with 50 ml polypropylene centrifuge tubes ( see Note 7 ).
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8楼2014-08-05 00:06:08
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yyc12596

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microvesicle的提取：

1. Transfer equal volume of the supernatant (collected in step 10
in Subheading 3.1 ) to 3 ml fresh ultracentrifuge tubes ( see
Note 8 ).
2. Mark the upper side of each ultracentrifuge tube with a waterproof
mark.
3. Put ultracentrifuge tubes in the fi xed-angle rotor of ultracentrifuge
with tweezers and make the waterproof mark face up.
4. Centrifuge for 2 h at 100,000 × g , 4 °C ( see Note 9 ).
5. Carefully take each ultracentrifuge tube out with tweezer ( see
Note 10 ).
6. Note the position of microvesicle pellet and remove the supernatant
completely (do not discard it) without disturbing the
pellet ( see Note 11 ).
7. Collect the supernatant in a 50-ml polypropylene centrifuge
tubes for later use.
8. Resuspend the pellet in each tube serially in 1 ml of PBS using
pipetman ( see Note 12 ).
9. Pool the resuspended pellets from all the tubes containing
urine from one subject in a single centrifuge tube.
10. Add appropriate volume of PBS if the volume of the collected
pellet solution is less than three-quarters of the whole volume
of the tube. Note the fi nal volume.
11. Make the waterproof mark on the side of the tube that contains
sample.
12. Add equal volume of PBS to one other ultracentrifuge tube.
It is used as a balancing tube.
13. Symmetrically put the two tubes in the fi xed-angle rotor of
ultracentrifuge with tweezers and make the mark of the tube
with sample face up.
14. Centrifuge for 2 h at 100,000 × g , 4 °C.
15. Carefully take each ultracentrifuge tube out with tweezers.
16. Note the position of microvesicle pellet and remove the supernatant
completely without disturbing the pellet ( see Note 13 ).
17. Add 300 μ l of guanidine thiocyanate containing lysis buffer to
the microvesicle pellet ( see Note 14 ).
18. Store microvesicle at −80 °C and avoid repeated freezing and
thawing.
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9楼2014-08-05 00:07:55
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yyc12596

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两个protocol很相似，基本就是三步：
  1. 1000g（3000g）把死细胞和细胞碎片离心下来，保留上清；
  2. 10000g把大的vesicle离心下来，保留上清。
  3. 0.22uM把上清过滤一下，然后超声离心，100000g 2h（2.5h），得到的沉淀就是microvesicle.

   先自己做做看，会什么情况。
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10楼2014-08-05 00:11:18
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