请老师帮忙分析网织血小板流式结果

tco_001

如题，做的网织血小板检测，采用CD41和TO双标记的方法。全血EDTA抗凝360g离心取上清得PRP，PRP加CD41和TO避光孵育15min后上机，首先使用FS和CD41找出血小板的population，即CD41+，然后在CD41+里面用TO和SS找出TO+的，得到网织血小板
现在审稿人有个问题，第一个图用FS和CD41找出血小板的population有5个群的细胞，想问一下各位老师除了划出来的血小板以后其他的cell cluster是什么啊？
还有审稿人说我第二个图用TO不足以区分TO和血小板的非特异性结合，我应该怎么做呢？

niwanmao

那些散在的应该都是碎片，评审可能不是搞流式的，所以会提出这种疑问，建议你可以采用下面这个FSC/SSC散点图先排除掉，再设CD41/FSC门。


至于TO这个图，建议你加个同型对照的图，他们会更容易承认。

tco_001

再次请假倪老师，我如果做同型对照应该怎么做呢？机器的门是工程师设置好的，一直没有调整过。是用什么染血小板然后上机呢？

niwanmao

tco_001 发表于 2014-12-18 15:14
再次请假倪老师，我如果做同型对照应该怎么做呢？机器的门是工程师设置好的，一直没有调整过。是用什么染血 ...

哦，不对，TO没有同型 对照，不好意思。
应该是空白对照，也就是TO这个通道（FL4）不染色的标本

tco_001

niwanmao 发表于 2014-12-18 15:41
哦，不对，TO没有同型 对照，不好意思。
应该是空白对照，也就是TO这个通道（FL4）不染色的标本 ...

那倪老师我根据您的意见再修改了一下您看这样的图片还有问题么？
Method: Percentage of Immature platelet fraction is the proportion of reticulated platelets within the total number of platelets. We conducted the analysis based on previous studies. In detail, EDTA anti-coagulated blood were centrifuged at 120g for 4 minutes. Then the supernatant, which is platelet rich plasma, was harvested. 10 ul of supernatant and 20ul CD41-PE antibody (BD, Biosciences) with 1ml thiazole orange (TO, Sigma-Aldrich) were mixed in the dark for 15 minutes. Analysis of the samples was performed with a flow cytometer (Beckman-Coulter, Miami, FL, USA). The platelet population in forward (versus side) scattering was confirmed as the population of CD41-PE positive events in the cytogram and was gated for this platelet population area. Based on RNA staining by TO, TO positivity was made by measuring both forward light scatter and green (540 nm) fluorescence using logarithmic amplification among CD41 positive events.

Figure legend: Gating strategies for IPF% analysis. Selected patients from non-survivor group (figure A and B) and survivor group (figure C and D) were demonstrated. Figure A and Figure C showed the FSC/CD41-PE plot for platelets. Figure B and Figure D showed the gating of CD41+ cells. The CD41+ population was backgated into the TO/SSC plot.CD41+TO+ platelets with appropriated size were viewed as IPF

tco_001

图片重新发一楼吧

niwanmao

tco_001 发表于 2014-12-18 21:21
图片重新发一楼吧

结果可以啊。

tco_001

niwanmao 发表于 2014-12-18 21:41
结果可以啊。

主要就是这个结果被审稿人提了如下的问题：Figure 1: in the FS/FL2 scatterplots A and C four or five cell clusters are visible. What do they represent? Figures B and D seem to suggest that TO positivity is defined using the FL4 signal only. This is absolutely insufficient for correcting size-dependent non-specific uptake of TO by platelets.
前一个问题已经解决了，但是后一个不知道要点是什么，所以就不明白了。是我method或者figure legend没有写清楚么倪老师？

niwanmao

tco_001 发表于 2014-12-18 22:12
主要就是这个结果被审稿人提了如下的问题：Figure 1: in the FS/FL2 scatterplots A and C four or five  ...

后一个问题我估计是评审搞乱了，你其实已经用TO和SSC设门了，要么你改成TO和FSC设门，然后再加一管未染色的试试。

tco_001

niwanmao 发表于 2014-12-19 09:51
后一个问题我估计是评审搞乱了，你其实已经用TO和SSC设门了，要么你改成TO和FSC设门，然后再加一管未染色 ...

我想评审的意见是不是要我去define TO positivity. 我还请教了我们一个再国外学习的同学，他们实验室回复If this was done on a one-dimensional fluorescence signal only, the method would be inadequate, as platelets display significant volume-dependent background staining with TO that is not related to their RNA content.但是我看了一下文献，TO+不就是one-dimensional fluorescence signal去定义的么。。。。。难道还有其他方法？