Data_Sheet_1_Sizing Up Extracellular DNA: Instant Chromatin Discharge From Cells When Placed in Serum-Free Conditions.pdf

How do you wash cells? Three out of four of our colleagues use experimental procedures during everyday lab-bench work that can severely impair data interpretation depending on how cells are handled. We show here that a subpopulation (2–3%) of human leukocytes immediately induce a yet unclassified lytic cell death, concomitant with discharge of chromatin entities and cell elimination, when placed in protein-free solutions (i.e., PBS and HBSS). DNA release was not restricted to hematopoietic cells but occurred also in HEK293T cells. Albumin, fetal bovine serum, polyethylene glycol, and Pluronic F-68 supplements prevented chromatin discharge. Expelled chromatin was devoid of surrounding membranes but maintained its original nuclear shape, although ∼10 times enlarged. These structures differed from DNA appearance after osmotic or detergent-induced cell lysis. Besides sounding a cautionary note to the neutrophil extracellular trap (NET) research community, in which ∼50% of all published studies used protein-free media for NET-formation, our study also provides a rapid tool for analysis of chromatin organization.

如何清洗细胞？在我们日常的实验室操作中，有四分之三的同事使用实验程序，这些程序可能会根据细胞处理方式的不同而严重损害数据解读。本研究揭示，当人类白细胞置于无蛋白溶液（例如磷酸盐缓冲盐溶液（PBS）和乙二胺四乙酸（HBSS））中时，会出现一个亚群（2-3%）的细胞立即诱导一种尚未分类的溶解性细胞死亡，伴随染色质实体和细胞的释放。DNA的释放并不仅限于造血细胞，也发生在HEK293T细胞中。白蛋白、胎牛血清、聚乙二醇和普鲁兰F-68的补充剂可防止染色质释放。被排出的染色质缺乏周围的膜结构，但保持了其原始的核形状，尽管体积扩大了约10倍。这些结构与渗透压或洗涤剂诱导的细胞溶解后的DNA外观不同。除了对中性粒细胞胞外陷阱（NET）研究领域的警示外，该领域约50%的已发表研究使用了无蛋白培养基进行NET形成，我们的研究还提供了一种快速分析染色质组织的工具。