title: Correlative analyses of nitric oxide generation rates and nitric oxide synthase levels in individual cells using a modular cell-retaining device.
abstract: Nitric oxide (NO) is recognized as one of the major immune system agents involved in the pathogenesis and control of various diseases that may benefit from novel drug development, by exploiting NO signaling pathways and targets. This calls for detection of both intracellular levels of NO and expression of its synthesizing enzymes (NOS) in individual, intact, living cells. Such measurements are challenging, however, due to short half-life, low and fluctuating concentrations of NO, cellular heterogeneity, and inability to trace the same cells over time. The current study presents a device and methodology for correlative analysis of NO generation rates and NOS levels in the same individual cells, utilizing fluorescent imaging followed by immunohistochemistry (IHC). U937 promonocyte cell populations demonstrated significant heterogeneity in their baseline levels, in NO-generation kinetics, and in their response rates to stimuli. Individual cell analysis exposed cell subgroups which showed enhanced NO production upon stimulation, concomitantly with significant up-regulation of inducible NOS (iNOS) levels. Exogenous NO modulated the expression of iNOS in nondifferentiated cells within 1 h, in a dose-dependent manner, while treatment with lysophosphatidylcholine (LPC) enhanced the expression of iNOS, demonstrating a nondependence on NO production.
authors: The Biophysical Interdisciplinary Schottenstein Center for the Research and Technology of the Cellome, Bar Ilan University, Ramat Gan, 52900 Israel.
authors: Yana
authors: Y
authors: Shafran
authors: Naomi
authors: N
authors: Zurgil
authors: Elena
authors: E
authors: Afrimzon
authors: Yishay
authors: Y
authors: Tauber
authors: Maria
authors: M
authors: Sobolev
authors: Asher
authors: A
authors: Shainberg
authors: Mordechai
authors: M
authors: Deutsch
mesh_terms: D002460:Cell Line; D019793:Fluorescein / Q000737:chemistry; D005456:Fluorescent Dyes / Q000737:chemistry; D005978:Glutathione / Q000378:metabolism; D006801:Humans; D007150:Immunohistochemistry; D008244:Lysophosphatidylcholines / Q000494:pharmacology; D018613:Microscopy, Confocal; D009569:Nitric Oxide / Q000378:metabolism*; D052247:Nitric Oxide Synthase Type II / Q000378:metabolism*; D015854:Up-Regulation / Q000187:drug effects
chemical_list: C414689:4,5-diaminofluorescein diacetate; D005456:Fluorescent Dyes; D008244:Lysophosphatidylcholines; D009569:Nitric Oxide; D052247:Nitric Oxide Synthase Type II; D005978:Glutathione; D019793:Fluorescein