如何选择FSC-A vs. FSC-H和FSC-W vs. FSC-H等坐标轴去除粘连细胞。

immunology

今天看了个文章，讲的是去除粘连细胞的时候，有的选择的是FSC-A vs. FSC-H，有的选择的是FSC-W vs. FSC-H，还有的选的是SSC-A vs. SSC-H或者SSC-W vs. SSC-H，到底应该怎么选择。这篇文章这么说的The basics of doublet discrimination is to detect disproportions between cell size vs. cell signal. Digital instruments have a function that allows one to correlate A and H so that they present the same variation when PMT voltages are changed, so that they can be correlated one to the other. In BD instruments we call this Area Scaling. When this factor is set properly, even though the voltage pulse is different between A and H, they will have the same reported value (that will be used to represent this particular event in the dot plot). So, if anyone has ever positioned the same parameter on both axis of a dot plot, you'll know that the graph that will apear is a perfect diagonal line (45 degrees, passing through ZERO). So, the basics of A vs. H strategy is, assuming that the instrument is set correctly and that A equals H, when correlating the same parameter (for instance, FSC) A vs. H, the same cell will have the same (or very similar) value in both axis. Therefore, all singlet events will be presented in a more diagonal display than doublets.However here is a thing that very few people no: the only way you can correctly discriminate doublets using the FSC-H vs. FSC-A strategy is ensuring that FSC Area Scaling has been set correctly (not with beads, but with the cells from your experiment). If this has not been done, there is a chance of error.Using the H vs. W strategy though is not affected by Area Scaling since both parameters are independent one from another. Therefore, it tends to be the more accurate way of performing doublet discrimination.
我翻译了一下：粘连体辨别的基本原理是检测细胞大小和细胞信号之间的比例失调。数字型流式仪有一个功能，可以使用一个参数把a和H联系起来，这样当PMT电压发生变化时，它们就会呈现相同的变化，这样它们就可以相互关联。在BD仪器中，我们称之为面积缩放Area Scaling。此参数设置妥当时，即使A和H的电压脉冲不同，他们也会有相同的报告值。如果在点图的X轴和Y轴放置相同的参数，图像肯定是过原点斜率为45°的对角线，所以，A vs. H的设置的策略的基本原理是，因此，A vs. H策略的基本原理是，假设仪器设置正确，A等于H，当将相同的参数(例如FSC) A与H关联时，相同的细胞在两个轴上都有相同(或非常相似)的值。（北京大学基因中心）因此，所有单细胞events都将比粘连细胞的events显示在更接近对角线处。
值得注意的事，用FSC-H和FSC-A策略区分单细胞时，确保用实验细胞而不是微球等去调整正确的FSC Area Scaling。
当用H vs. W区分单细胞时，因为两者都是彼此独立的，均不受Scaling的影响，所以推荐用H vs. W区分单细胞。


总结，能用FSC-W vs. FSC-H去除双联体细胞，就不用FSC-A vs. FSC-H；SSC与之相同。


下面放几个不同坐标轴怎么区分单细胞的图：






如果用H和W区分单细胞的话，选择W较小的群，因为W直接反映了细胞通过的时间，时间越长表示粘连细胞的可能性越大；
如果用A和H区分单细胞的话，首先调整Scaling使大部分细胞群到对角线上，然后圈在H相同的情况下，A较小的一群。因为H相同，A越大，W越大，粘连细胞可能性越大。
如果用A和W区分单细胞的话，选择W较小的群，道理同第一条。




注：以上英文部分摘自公众号实验小白。   请批评指正。

niwanmao

从原理上说，H是肯定不变，可能大家纠结的是用W还是A的问题，实际上这两个不是每个机器都有的，W我记得可能只有BD的机器有，所以用A的人会更多一些。
至于上面所说的缩放因子，这是也是BD的机器特有的，调整之后，确实会使区分能力更强。