He Chang, Zhi Chen, Li Gao, Hong Cao, Yongqiang Wang, Shijun J Zheng
{"title":"用流式细胞仪检测嗜热症。","authors":"He Chang, Zhi Chen, Li Gao, Hong Cao, Yongqiang Wang, Shijun J Zheng","doi":"10.3791/66912","DOIUrl":null,"url":null,"abstract":"<p><p>Pyroptosis is an inflammatory type of programmed cell death predominantly driven by the formation of plasma membrane pores by the N-terminus generated from the cleaved Gasdermin (GSDM) family proteins. Examination of membrane-attached GSDM-<sup>NT</sup> by Western Blot is the most commonly used method for evaluating pyroptosis. However, it is difficult to differentiate cells with pyroptosis from other forms of cell death using this method. In this study, Infectious Bursal Disease Virus (IBDV)-infected DF-1 cells were employed as a model to quantify the proportion of cells undergoing pyroptosis by flow cytometry, utilizing specific antibodies against the N-terminal fragment of chicken GSDME (chGSDME-<sup>NT</sup>) and propidium iodide (PI) staining. The chGSDME-<sup>NT</sup>-positive cells were readily detectable by flow cytometry using Alexa Fluor 647-labeled anti-chGSDME-<sup>NT</sup> antibodies. Moreover, the proportion of chGSDME-<sup>NT</sup>/PI double-positive cells in IBDV-infected cells (around 33%) was significantly greater than in mock-infected controls (P < 0.001). These findings indicate that examination of membrane-bound chGSDME-<sup>NT</sup> by flow cytometry is an effective approach for determining pyroptotic cells among cells undergoing cell death.</p>","PeriodicalId":48787,"journal":{"name":"Jove-Journal of Visualized Experiments","volume":null,"pages":null},"PeriodicalIF":1.2000,"publicationDate":"2024-05-31","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":"{\"title\":\"Examination of Pyroptosis by Flow Cytometry.\",\"authors\":\"He Chang, Zhi Chen, Li Gao, Hong Cao, Yongqiang Wang, Shijun J Zheng\",\"doi\":\"10.3791/66912\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<p><p>Pyroptosis is an inflammatory type of programmed cell death predominantly driven by the formation of plasma membrane pores by the N-terminus generated from the cleaved Gasdermin (GSDM) family proteins. Examination of membrane-attached GSDM-<sup>NT</sup> by Western Blot is the most commonly used method for evaluating pyroptosis. However, it is difficult to differentiate cells with pyroptosis from other forms of cell death using this method. In this study, Infectious Bursal Disease Virus (IBDV)-infected DF-1 cells were employed as a model to quantify the proportion of cells undergoing pyroptosis by flow cytometry, utilizing specific antibodies against the N-terminal fragment of chicken GSDME (chGSDME-<sup>NT</sup>) and propidium iodide (PI) staining. The chGSDME-<sup>NT</sup>-positive cells were readily detectable by flow cytometry using Alexa Fluor 647-labeled anti-chGSDME-<sup>NT</sup> antibodies. Moreover, the proportion of chGSDME-<sup>NT</sup>/PI double-positive cells in IBDV-infected cells (around 33%) was significantly greater than in mock-infected controls (P < 0.001). These findings indicate that examination of membrane-bound chGSDME-<sup>NT</sup> by flow cytometry is an effective approach for determining pyroptotic cells among cells undergoing cell death.</p>\",\"PeriodicalId\":48787,\"journal\":{\"name\":\"Jove-Journal of Visualized Experiments\",\"volume\":null,\"pages\":null},\"PeriodicalIF\":1.2000,\"publicationDate\":\"2024-05-31\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Jove-Journal of Visualized Experiments\",\"FirstCategoryId\":\"103\",\"ListUrlMain\":\"https://doi.org/10.3791/66912\",\"RegionNum\":4,\"RegionCategory\":\"综合性期刊\",\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"\",\"PubModel\":\"\",\"JCR\":\"Q3\",\"JCRName\":\"MULTIDISCIPLINARY SCIENCES\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Jove-Journal of Visualized Experiments","FirstCategoryId":"103","ListUrlMain":"https://doi.org/10.3791/66912","RegionNum":4,"RegionCategory":"综合性期刊","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q3","JCRName":"MULTIDISCIPLINARY SCIENCES","Score":null,"Total":0}
Pyroptosis is an inflammatory type of programmed cell death predominantly driven by the formation of plasma membrane pores by the N-terminus generated from the cleaved Gasdermin (GSDM) family proteins. Examination of membrane-attached GSDM-NT by Western Blot is the most commonly used method for evaluating pyroptosis. However, it is difficult to differentiate cells with pyroptosis from other forms of cell death using this method. In this study, Infectious Bursal Disease Virus (IBDV)-infected DF-1 cells were employed as a model to quantify the proportion of cells undergoing pyroptosis by flow cytometry, utilizing specific antibodies against the N-terminal fragment of chicken GSDME (chGSDME-NT) and propidium iodide (PI) staining. The chGSDME-NT-positive cells were readily detectable by flow cytometry using Alexa Fluor 647-labeled anti-chGSDME-NT antibodies. Moreover, the proportion of chGSDME-NT/PI double-positive cells in IBDV-infected cells (around 33%) was significantly greater than in mock-infected controls (P < 0.001). These findings indicate that examination of membrane-bound chGSDME-NT by flow cytometry is an effective approach for determining pyroptotic cells among cells undergoing cell death.
期刊介绍:
JoVE, the Journal of Visualized Experiments, is the world''s first peer reviewed scientific video journal. Established in 2006, JoVE is devoted to publishing scientific research in a visual format to help researchers overcome two of the biggest challenges facing the scientific research community today; poor reproducibility and the time and labor intensive nature of learning new experimental techniques.