Department of Chemistry, University of North Carolina, Chapel Hill, NC 27599, USA.
Analyst. 2019 Jan 28;144(3):961-971. doi: 10.1039/c8an01353e.
Chemical cytometry using capillary electrophoresis (CE) is a powerful tool for measuring single-cell enzyme activity. However, these measurements are often confounding as dynamic processes within cells rapidly change depending on environment, meaning that cell handling, transport, and storage can affect signaling pathways and alter results. To meet these challenges, we describe a method utilizing aldehyde fixation to simultaneously terminate cellular reactions across a population, freezing reaction results in time prior to analytical analysis. Fluorescent sphingosine was loaded into cells of different lineages (leukemia and lymphoma cell lines and primary leukemia cells) and allowed to react before fixing. The remaining sphingosine and any products formed were then quantified with chemical cytometry utilizing CE. When cells were loaded with sphingosine followed by glyoxal fixation and immediate analysis, 55 ± 5% of lipid was recoverable compared to an unfixed control. Storage of fixed cells for 24 h showed no statistical differences in total amount of recoverable sphingolipid compared to samples analyzed immediately after fixation-though there was a difference in recovery of low-abundance products. Sphingosine kinase activity decreased in response to inhibitor treatment compared to treatment with a DMSO vehicle (21 ± 3% product formed in inhibitor-treated cells vs. 57 ± 2% in control cells), which was mirrored in single-cell measurements. This "fix and assay" strategy enables measurement of sphingosine kinase activity in single cells followed by subsequent analytical assay separated in space and time from reaction initiation, enabling greater temporal control over intracellular reactions and improving future compatibility with clinical workflow.
利用毛细管电泳（CE）进行化学细胞术是测量单细胞酶活性的强大工具。然而，这些测量通常会受到干扰，因为细胞内的动态过程会根据环境迅速变化，这意味着细胞处理、运输和储存会影响信号通路并改变结果。为了应对这些挑战，我们描述了一种利用醛固定化的方法，该方法可以同时终止群体中的细胞反应，在分析之前将反应结果冻结在时间上。将荧光神经酰胺加载到不同谱系的细胞（白血病和淋巴瘤细胞系和原代白血病细胞）中，并在固定之前让其反应。然后利用 CE 进行化学细胞术对剩余的神经酰胺和形成的任何产物进行定量。当细胞用神经酰胺加载，然后用乙二醛固定并立即分析时，与未经固定的对照相比，可回收的脂质为 55±5%。固定细胞储存 24 小时与固定后立即分析相比，可回收鞘脂的总量没有统计学差异 - 尽管在低丰度产物的回收方面存在差异。与用 DMSO 载体处理的细胞相比，抑制剂处理会导致鞘氨醇激酶活性降低（抑制剂处理的细胞中形成的产物为 21±3%，而对照细胞中为 57±2%），单细胞测量也反映了这一点。这种“固定和检测”策略能够测量单细胞中的鞘氨醇激酶活性，然后在空间和时间上与反应起始分离进行后续分析，从而对细胞内反应进行更大的时间控制，并提高与临床工作流程的未来兼容性。
