请问大家有用流式检测exosome的步骤吗？
请问大家有用流式检测exosome的步骤吗？可以共享一下吗？谢谢！1. Incubate 30 µg of exosomes or 30 µg BSA (negative control) in a 10 µl volume of beads at RT for 15 min with continuous rotation.
请问下这里exosomes和latex beads是什么原理结合的？ 同求，如何做呢，求大神指教 我正在用的，仅供参考
Flow cytometry analysis of exosomes-bound beads
1. Incubate 30 µg of exosomes or 30 µg BSA (negative control) in a 10 µl volume of beads at RT for 15 min with continuous rotation.
2. This suspension was diluted to 1ml with PBS and incubate the mixture at RT for a further 2 h or at 4 ℃ overnight with agitation.
3. Terminate the coupling reaction by the addition of 100 mM glycine and 2% BSA in PBS and left rotating for 30 min at RT.
4. Exosomes-bound beads were washed once in 2% BSA in PBS and centrifuged for 1 min at 14,800g, blocked with 10% BSA with rotation at room temperature for 30 min, washed a second time in 2 % BSA and centrifuged for 1 min at 14,800g, and incubated with anti-X during 30 min rotating at 4 ℃. .
5. Beads were centrifuged for 1 min at 14,800g, the supernatant was discarded and beads were washed in 2% BSA and centrifuged for 1 min at 14,800g.
6. Alexa-488-tagged secondary antibodies (Life Technologies, 3 ml of antibody in 20 ml of 2 % BSA) were used during 30 min with rotation at 4 ℃. Secondary antibody incubation alone was used as control and to gate the beads with X＋-bound exosomes. The percentage of positive beads was calculated relative to the total number of beads analysed per sample (100,000 events). This percentage was therein referred to as the percentage of beads with X＋ exosomes.
我们的beads是4um的，Thermo家的 luckkit 发表于 2015-10-28 14:44
我正在用的，仅供参考
Flow cytometry analysis of exosomes-bound beads
能求一份文献出处么？..... 本帖最后由 子非鱼 于 2016-1-26 14:39 编辑
weaver 发表于 2015-11-4 09:49
能求一份文献出处么？.....
有个类似的：Sonia A. Melo, Linda B. Luecke, Christoph Kahlert et al. Glypican-1 identifies cancer exosomes and detects early pancreatic cancer. nature. doi:10.1038/nature14581.
luckkit 发表于 2015-10-29 13:17
我们的beads是4um的，Thermo家的
luckkit同学，你已经做过exosome的磁珠分选吗？效果怎么样？必需搭配他们家的抗体吗？我从Abcam买的抗体CD63可用作流式，不知道可不可以用？我看他们家的珠子基本都是15ml包装的，可以分选多少外泌体啊？求分享~~~ luckkit 发表于 2015-10-29 13:17
我们的beads是4um的，Thermo家的
可以受累告知一下货号吗，新手小白不知怎么选择。不胜感激。 真的是头都大了