以流式细胞术同时检测caspase-1与碘化丙啶或AnnexinV分析小鼠骨髓巨噬细胞的炎性死亡

Establishment of a flow cytometry-based test for the detection of pyroptosis of BMDM

目的明确以流式细胞术同时检测caspase-1与碘化丙啶或AnnexinV的方法测定小鼠骨髓巨噬细胞炎性死亡的可行性。方法从野生型（wildtype，WT）C57BL／6小鼠和／或C57BL／6背景的easpase．1-/-小鼠分离骨髓细胞，巨噬细胞集落刺激因子（macrophagecolony—stimulatingfactor，M—CSF）刺激分化7d成为骨髓巨噬细胞后，分别进行磷酸盐缓冲液（PBS）、脂多糖（LPS）、LPS＋ATP（三磷酸腺苷）处理。蛋白印迹法（Westernblot）和酶联免疫吸附试验（enzyme—linkedimmunosorbentassay，ELISA）检测细胞和上清中的IL-1β和caspase-1的切割成熟及分泌情况；乳酸脱氢酶（1actatedehydrogenase，LDH）检测试剂盒检测细胞上清LDH的释放量；以3种流式细胞染色法：TMRred＋caspase-1、PI（propidiumiodide）＋easpase-1和AnnexinV＋caspase-1检测小鼠骨髓巨噬细胞的炎性死亡。结果LPS＋ATP处理后，NLRP3炎症小体被激活（细胞和上清中有IL．1B和caspase-1的切割成熟和分泌）；wT即C57BL／6组细胞上清LDH的释放量多于caspase-1-/-组，表明诱导发生的细胞死亡中存在依赖于easpase-1的炎性死亡方式；流式细胞术同时检测caspase-1与PI或AnnexinV，结果显示LPS＋ATP处理组炎性死亡的百分比显著高于PBS组和LPS组，与已报道的流式检测caspase-1＋TMRred方法的结果类似。结论流式细胞术同时检测caspase-1与PI或AnnexinV的方法可用于确定小鼠骨髓巨噬细胞的炎性死亡。与已有的流式检测easpase-1＋TMRred方法一样，这两种基于流式细胞术的炎性死亡的检测方法，为小鼠骨髓巨噬细胞的炎性死亡提供了更加直观精准的检测手段。

Objective To establish a flow cytometry-based test for the detection of pyroptosis of murine bone marrow-derived macrophages （BMDM）. Methods Bone marrow cells were isolated from wild type （WT） C57BL/6 mice and/or caspase-1-/- C57BL/6 mice and then stimulated with macrophage colonystimulating factor （M-CSF） to differentiate into murine BMDM. PBS, LPS and LPS＋adenosine triphosphate （ATP） were respectively used to stimulate the BMDM. Western blot assay was performed to detect the cleav- age of IL-1β and caspase-1. The levels of IL-1β in the supernatants of cell culture were measured by ELISA. Lactate dehydrogenase （LDH） released in the culture media was detected by using LDH kit. The pyroptosis of murine BMDM was detected by using flow cytometry analysis after double-staining with TMR red ＋caspase-1, AnnexinV ＋caspase-1 and propidium iodide （PI）＋caspase-1. Results 1L-1β was detected in the culture medium of BMDM treated with LPS＋ATP and the cleavage of IL-1 β and caspase-1 was confirmed by Western blot assay, which indicated that the NLRP3 inflammasome was activated by LPS＋ATP treatment. Compared with the caspase-1-/- mice group, higher levels of LDH were detected in the culture medium of BMDM isolated form the WT mice. Results of the flow cytometry analysis after staining BMDM with caspase- 1 plus AnnexinV or PI showed that more cells undergoing pyroptosis were detected in the LPS＋ATP treat- ment group than that in LPS or PBS treatment group, which were consistent with the results of the reported flow cytometry with caspase-1 ＋TMR red staining. Conclusion The flow cytometry-based test with double-staining of caspase-1 plus AnnexinV or PI could be used for the detection of pyroptosis of murine BMDM.