Application of flow cytometry with a fluorescent dye to measurement of intracellular nitric oxide in plant cells

Abstract

Main conclusionA simple and rapid method involving flow cytometry and NO-specific probe (DAF-FM DA) proved useful for detection and determination of intracellular NO production in Medicago truncatula suspension cells and leaves as well as in cells of Avena fatua, Amaranthus retroflexus embryos and leaves.The measurement of nitric oxide (NO) in plant material is important for examining the regulatory roles of endogenous NO in various physiological processes. The possibility of detecting and determining intracellular NO production by flow cytometry (FCM) with 4-amino-5-methylamino-2′,7′-difluorofluorescein (DAF-FM DA), an NO-specific probe in Medicago truncatula cells in suspension and leaves as well as in cells of embryos and leaves of Avena fatua L. or Amaranthus retroflexus L. was explored. To detect and measure NO production by cell suspension or embryos and leaves, the recommended DAF-FM DA concentration is 5 or 10 µM, respectively, applied for 30 min. Exogenous NO increased the intensity of the fluorescent signal in embryos and leaves of both plants, while carboxy-PTIO (cPTIO), an NO scavenger, decreased it. Thus, these results demonstrate that NO can be detected and an increase and a decrease of its intracellular level can be estimated. Wounding was observed to increase the fluorescence signal, indicating an increase in the intracellular NO level. In addition, the levels of exogenous and endogenous ascorbic acid were demonstrated to have no effect on the NO-related fluorescence signal, indicating the signal’s specificity only in relation with NO. The applicability of the proposed method for detection and determination of NO was confirmed (1) by in situ NO imaging in cell suspensions and (2) by determining the NO concentration in embryos and leaves using the Griess reagent. In view of the data obtained, FCM is recommended as a rapid and simple method with which to detect and determine intracellular NO production in plant cells.

Manoalide Shows Mutual Interaction between Cellular and Mitochondrial Reactive Species with Apoptosis in Oral Cancer Cells

“…After manoalide treatment for 6 h, cells were stained with DCFH-DA [ 26 ] (1 mM, 30 min), DHE [ 27 ] (5 μ M, 30 min), MitoSOX Red [ 28 ] (0.25 mM, 10 min), DAF-FM [ 29 ] (10 μ M, 30 min), or HPF [ 30 ] (10 μ M, 30 min), and washed with 1x PBS. Subsequently, DCFH-DA, DAF-FM, and HPF were observed by Guava® easyCyte flow cytometer (Merck KGaA; Darmstadt, Germany) at ex/em for 488/525 nm.…”

2-Methoxyestradiol and Its Combination with a Natural Compound, Ferulic Acid, Induces Melanoma Cell Death via Downregulation of Hsp60 and Hsp90

“…We have previously evidenced that one of the anticancer modes of 2-ME is associated with a selective increase in the nitric oxide level [8,9,46,47]. erefore, in the next part of the study, we aimed to determine the impact of compounds on changes within the level of nitric oxide in melanoma A375 cells via DAF-DA staining [8,52,53]. Notably, herein, we evidenced that induction of nitric oxide after 8-hour treatment with 10 μM 2-ME can be also extended to the melanoma A375 cellular model (Figure 4(b)).…”

Present knowledge and controversies, deficiencies, and misconceptions on nitric oxide synthesis, sensing, and signaling in plants

“…The community working in different aspects of NO‐related biology faces a systematic bottleneck in trying to quantify the endogenous levels of NO. Fluoresceins, such as diaminofluorescein (DAF), have been extensively used to detect NO (Foissner, Wendehenne, Langebartels, & Durner, ) and in combination with flow cytometry allow quantifying NO in cell suspensions (Kępczyński & Cembrowska‐Lech, ). However, DAF does not bind directly to NO but to N 2 O 3 , which is an oxidation product of NO (Kojima et al .…”