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yyc12596新虫 (著名写手)
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microvesicle ultracentrafuge 已有1人参与
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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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yyc12596
新虫 (著名写手)
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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
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
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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/ |
2楼2014-08-04 23:31:26
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. |
3楼2014-08-04 23:34:31
yyc12596
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4楼2014-08-04 23:35:51
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. |
5楼2014-08-04 23:43:28
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 |
6楼2014-08-04 23:44:47
yyc12596
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7楼2014-08-05 00:03:59
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 ). |
8楼2014-08-05 00:06:08
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. |
9楼2014-08-05 00:07:55
yyc12596
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10楼2014-08-05 00:11:18
