碘化丙啶
溶解
二氯荧光素
流式细胞术
分子生物学
呼吸爆发
罗丹明123
活力测定
化学
生物
生物化学
细胞内
体外
细胞凋亡
程序性细胞死亡
多重耐药
抗生素
作者
Juha Vuorte,Sten-Erik Jansson,Heikki Repo
出处
期刊:Cytometry
[Wiley]
日期:2001-04-01
卷期号:43 (4): 290-296
被引量:50
标识
DOI:10.1002/1097-0320(20010401)43:4<290::aid-cyto1061>3.0.co;2-x
摘要
Background Neutrophil subpopulations with enhanced oxidative reactivity have been described in a number of clinical and in vitro settings. In the dichlorofluorescin (DCFH) oxidation assay, it is essential to maintain cellular viability and plasma membrane integrity through all stages of sample preparation. The process of erythrocyte lysing is crucial because a number of commercial lysing reagents can increase leukocyte membrane permeability. Methods We assessed viability [propidium iodide (PI) method], DCFH oxidation, and CD11b expression of resting or in vitro–stimulated neutrophils exposed to six different red cell lysing procedures. Results Formaldehyde-containing reagents (Optilyse B, FACS Lyse, and Erythrolyse) but not hypotonic shock or ammonium chloride (NH4Cl) solutions rendered 91.4–99.8% of resting neutrophils PI positive, with concomitant reductions in dichlorofluorescein (DCF) fluorescence, suggesting efflux of the fluorochrome. However, when stimulated with N-formyl-methionyl-leucyl-phenylalanine or Yersinia enterocolitica and then treated with FACS Lyse or Erythrolyse, up to 69.9% of neutrophils remained PI negative and exhibited enhanced DCF fluorescence. CD11b expression of PI-positive and -negative neutrophils was comparable, suggesting that they were activated equally. Conclusions FACS Lyse and Erythrolyse can modify neutrophil plasma membrane integrity, whereas hypotonic shock and NH4Cl solutions retain cellular viability and are lysing methods of choice in evaluation of neutrophil respiratory burst by DCFH oxidation assay. Cytometry 43:290–296, 2001. © 2001 Wiley-Liss, Inc.
科研通智能强力驱动
Strongly Powered by AbleSci AI