Flow Cytometry Protocols for Surface and Intracellular Antigen Analyses of Neural Cell Types

Menon, Vishal; Thomas, Ria; Ar, Ghale; Reinhard, Christina; Pruszak, Jan

doi:10.3791/52241

Cited by 33 publications

(19 citation statements)

References 69 publications

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“…Analysis of the expression of cyclin D1 and cyclin D2 (sc-20044 PE, sc-53637 PE; Santa Cruz Biotechnology Inc.) in melanoma cell lines was carried out using Flow cytometry (Gallios; Beckman Coulter) 35 , 36 . The Per Fix-nc kit (Cat #B3116; Beckman Coulter) was used for fixing, permeabilizing, and preparing the cells for the assay.…”

Section: Methodsmentioning

confidence: 99%

A combination of high dose rate (10X FFF/2400 MU/min/10 MV X-rays) and total low dose (0.5 Gy) induces a higher rate of apoptosis in melanoma cells in vitro and superior preservation of normal melanocytes

et al. 2015

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The aim of this study was to determine the apoptotic effects, toxicity, and radiosensitization of total low dose irradiation delivered at a high dose rate in vitro to melanoma cells, normal human epidermal melanocytes (HEM), or normal human dermal fibroblasts (HDF) and to study the effect of mitochondrial inhibition in combination with radiation to enhance apoptosis in melanoma cells. Cells irradiated using 10X flattening filter-free (FFF) 10 MV X-rays at a dose rate of 400 or 2400 MU/min and a total dose of 0.25–8 Gy were analyzed by cell/colony counting, MitoTracker, MTT, and DNA-damage assays, as well as by quantitative real-time reverse transcriptase PCR in the presence or absence of mitochondrial respiration inhibitors. A dose rate of 2400 MU/min killed on average five-fold more melanoma cells than a dose rate 400 MU/min at a total dose of 0.5 Gy and preserved 80% survival of HEM and 90% survival of HDF. Increased apoptosis at the 2400 MU/min dose rate is mediated by greater DNA damage, reduced cell proliferation, upregulation of apoptotic genes, and downregulation of cell cycle genes. HEM and HDF were relatively unharmed at 2400 MU/min. Radiation induced upregulation of mitochondrial respiration in both normal and cancer cells, and blocking the respiration with inhibitors enhanced apoptosis only in melanoma cells. A high dose rate with a low total dose (2400 MU/min, 0.5 Gy/10X FFF 10 MV X-rays) enhances radiosensitivity of melanoma cells while reducing radiotoxicity toward HEM and HDF. Selective cytotoxicity of melanoma cells is increased by blocking mitochondrial respiration.

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Section: Methodsmentioning

confidence: 99%

A combination of high dose rate (10X FFF/2400 MU/min/10 MV X-rays) and total low dose (0.5 Gy) induces a higher rate of apoptosis in melanoma cells in vitro and superior preservation of normal melanocytes

et al. 2015

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“…Не так давно выдвигалось предположение, что эпендимоциты являются нейральными стволовыми клетками (НСК) [21]. Например, эпендимальные клетки обладают некоторыми фенотипическими характеристиками НСК -экспрессируют маркеры, характерные для НСК, такие как интегрин-бета 1, CD24, CD133 и Sox2 [22][23][24]. Однако, учитывая, что эпендимоциты все же имеют определенные особенности пролиферативной активности в постнатальном периоде в физиологических условиях и не полностью соответствуют критериям стволовых клеток, вопрос о принадлежности эпендимоцитов к НСК был закрыт [25,26].…”

Section: Review Articles фундаментальная и клиническая медицина ®unclassified

Brain ependymocytes in neurogenesis and maintaining integrity of blood-cerebrospinal fluid barrier

Успенская¹,

Моргун²,

Осипова³

et al. 2019

Fundamentalʹnaâ i kliničeskaâ medicina

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Here we review the physiology of brain ependymocytes which produce cerebrospinal fluid, regulate neurogenic niches, and contribute to neurogenesis in health and disease. We particularly focus on cilia as these organelles are pivotal to ensure the normal functioning of ependymocytes. The functional activity of ependymocytes is largely defined by their localisation in the central nervous system. Further studies of ependymal cell biology are required to better understand the mechanisms of neurological disorders and to discover novel therapeutic strategies aimed at correcting neurodegeneration and aberrant development of the brain.

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“…Following step 3.8, homogenize unfixed hindbrains to produce a single cell suspension for flow cytometry applications 15 .…”

Section: Protocolmentioning

confidence: 99%

The Mouse Hindbrain As a Model for Studying Embryonic Neurogenesis

Tata

Ruhrberg

2018

JoVE

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The mouse embryo forebrain is the most commonly employed system for studying mammalian neurogenesis during development. However, the highly folded forebrain neuroepithelium is not amenable to wholemount analysis to examine organ-wide neurogenesis patterns. Moreover, defining the mechanisms of forebrain neurogenesis is not necessarily predictive of neurogenesis in other parts of the brain; for example, due to the presence of forebrain-specific progenitor subtypes. The mouse hindbrain provides an alternative model for studying embryonic neurogenesis that is amenable to wholemount analysis, as well as tissue sections to observe the spatiotemporal distribution and behavior of neural progenitors. Moreover, it is easily dissected for other downstream applications, such as cell isolation or molecular biology analysis. As the mouse hindbrain can be readily analyzed in the vast number of cell lineage reporter and mutant mouse strains that have become available, it offers a powerful model for studying the cellular and molecular mechanisms of developmental neurogenesis in a mammalian organism. Here, we present a simple and quick method to use the mouse embryo hindbrain for analyzing mammalian neural progenitor cell (NPC) behavior in wholemount preparations and tissue sections.

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Flow Cytometry Protocols for Surface and Intracellular Antigen Analyses of Neural Cell Types

Cited by 33 publications

References 69 publications

A combination of high dose rate (10X FFF/2400 MU/min/10 MV X-rays) and total low dose (0.5 Gy) induces a higher rate of apoptosis in melanoma cells in vitro and superior preservation of normal melanocytes

A combination of high dose rate (10X FFF/2400 MU/min/10 MV X-rays) and total low dose (0.5 Gy) induces a higher rate of apoptosis in melanoma cells in vitro and superior preservation of normal melanocytes

Brain ependymocytes in neurogenesis and maintaining integrity of blood-cerebrospinal fluid barrier

The Mouse Hindbrain As a Model for Studying Embryonic Neurogenesis

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