Shared Research Resources, UTHealth, The University of Texas Health Science Center at Houston, Office of the Executive Vice President Academic and Research Affairs 1825 Pressler St 330G Houston, TX 77030, USA.
URMC Shared Resource Laboratories, URMC Flow Cytometry Facility, Medical Center 3-415, Rochester, NY 14642, USA.
Methods. 2018 Feb 1;134-135:130-135. doi: 10.1016/j.ymeth.2018.01.006. Epub 2018 Jan 31.
With the release and use of the Becton Dickenson FACS Diva Software, the use of Area as the default parameter came into play. As such, the use of area as a calculated parameter, methods were needed to be employed to ensure doublet discrimination and proper display on standard FSC/SSC. Improper setting of forward area scaling can alter the display cell populations. This combined with improper area gating strategy can lead to doublet inclusion which in sorting rare events can compromise sort purity. In extreme cases where area scaling with the individual lasers is ignored, differences can exist between Area and Height where compensation will likely not be optimal, particularly if one parameter - usually height is saturated. In addition, area scaling can impact population grouping. As FSC and individual laser area scaling is a function of event size, the most common error is to accept the setting determined by CS&T, which are 3.2 µm particles and proceed with the sample(s) without regard to the sample's actual size. With cellular events smaller or more likely larger than the CS&T beads, this will make the area scaling settings less than optimal. Analysis and sorting rare events with populations larger than the CS&T beads can be compromised if adjustments in FSC area scaling are not addressed. Proper FSC and laser area scaling must be determined empirically for each sample. Examples of the effects of sample size on area scaling will be presented in addition to gating and templates for determining area scaling.
随着 Becton Dickenson FACS Diva 软件的发布和使用，面积默认参数的使用开始发挥作用。因此，需要采用方法来使用面积作为计算参数，以确保二聚体的区分和在标准 FSC/SSC 上的正确显示。前向面积比例设置不当会改变显示细胞群。这与不当的面积门控策略相结合，可能导致二聚体的包含，在稀有事件的排序中，这可能会影响纯度。在面积与个别激光器的单独比例设置被忽略的极端情况下，可能会出现面积和高度之间的差异，补偿可能不会最佳，特别是如果一个参数——通常是高度——饱和。此外，面积比例会影响群体分组。由于 FSC 和单个激光的面积比例是事件大小的函数，最常见的错误是接受 CS&T 确定的设置，即 3.2µm 粒子，而不考虑样品的实际大小，就对样品进行处理。对于比 CS&T 珠子小或更可能大的细胞事件，这将使面积比例设置不够理想。如果不解决 FSC 面积比例调整问题，分析和排序大于 CS&T 珠子的稀有群体可能会受到影响。必须根据每个样品的实际情况经验性地确定 FSC 和激光的面积比例。除了门控和模板外，还将展示样品大小对面积比例的影响的例子，以确定面积比例。
