Cryopreservation of primary neurons for tissue culture

Kawamoto, Jerrolynn C.; Barrett, John N.

doi:10.1016/0006-8993(86)91222-9

Cited by 158 publications

(85 citation statements)

References 8 publications

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“…[9][10][11] Cortical tissue was disrupted by gentile trituration and seeded onto poly-L-lysinecoated dishes at a density of B2 Â 10 6 cells per well of a standard 6-well cell culture plate. Neuronal cultures were maintained for 7 days in N5 medium 12 with 5% (1 mg/mL protein) of an acid stable fraction of horse serum. 13 Cell cultures were then treated with different doses of poly(dA:dT) complexed with LyoVec, a cationic lipid-based transfection reagent, ranging from 2.5 to 20 mg/mL (Invivogen, San Diego, CA, USA) for 3 hours.…”

Section: Cell Culture and Inflammasome Stimulationmentioning

confidence: 99%

Pyroptotic Neuronal Cell Death Mediated by the AIM2 Inflammasome

Adamczak

Vaccari

Dale

et al. 2014

J Cereb Blood Flow Metab

252

237

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The central nervous system (CNS) is an active participant in the innate immune response to infection and injury. In these studies, we show embryonic cortical neurons express a functional, deoxyribonucleic acid (DNA)-responsive, absent in melanoma 2 (AIM2) inflammasome that activates caspase-1. Neurons undergo pyroptosis, a proinflammatory cell death mechanism characterized by the following: (a) oligomerization of apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC); (b) caspase-1 dependency; (c) formation of discrete pores in the plasma membrane; and (d) release of the inflammatory cytokine interleukin-1b (IL-1b). Probenecid and Brilliant Blue FCF, inhibitors of the pannexin1 channel, prevent AIM2 inflammasome-mediated cell death, identifying pannexin1 as a cell death effector during pyroptosis and probenecid as a novel pyroptosis inhibitor. Furthermore, we show activation of the AIM2 inflammasome in neurons by cerebrospinal fluid (CSF) from traumatic brain injury (TBI) patients and oligomerization of ASC. These findings suggest neuronal pyroptosis is an important cell death mechanism during CNS infection and injury that may be attenuated by probenecid.

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Section: Cell Culture and Inflammasome Stimulationmentioning

confidence: 99%

Pyroptotic Neuronal Cell Death Mediated by the AIM2 Inflammasome

Adamczak

Vaccari

Dale

et al. 2014

J Cereb Blood Flow Metab

252

237

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“…Cortical tissue was disrupted into a cell suspension by gentle trituration and seeded in 60-mm dishes at a density of 2 ϫ 10 6 cells/dish. Neurons were grown on poly-Llysine-coated tissue culture dishes in N5 medium that contained 5% serum fraction (24). Neurons were maintained for 12 days, and the neuronal nature of the majority of cells (95%) was confirmed electrophysiologically and immunohistochemically (22).…”

Section: Methodsmentioning

confidence: 99%

The Pannexin 1 Channel Activates the Inflammasome in Neurons and Astrocytes

Silverman¹,

Vaccari

Locovei³

et al. 2009

Journal of Biological Chemistry

491

603

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The inflammasome is a multiprotein complex involved in innate immunity. Activation of the inflammasome causes the processing and release of the cytokines interleukins 1␤ and 18. In primary macrophages, potassium ion flux and the membrane channel pannexin 1 have been suggested to play roles in inflammasome activation. However, the molecular mechanism(s) governing inflammasome signaling remains poorly defined, and it is undetermined whether these mechanisms apply to the central nervous system. Here we show that high extracellular potassium opens pannexin channels leading to caspase-1 activation in primary neurons and astrocytes. The effect of K ؉ on pannexin 1 channels was independent of membrane potential, suggesting that stimulation of inflammasome signaling was mediated by an allosteric effect. The activation of the inflammasome by K Pannexin 1 is a vertebrate ortholog of the invertebrate innexin gap junction proteins (1), but it does not appear to form functional gap junctions in vivo. Instead pannexin 1 acts as a membrane channel that carries ions and signaling molecules between the cytoplasm and the extracellular space (2, 3). As such, it is a candidate ATP release channel in various cell types, including erythrocytes, astrocytes, bronchial epithelial cells, and taste cells. Various functional roles have been ascribed to pannexin 1 including local vascular perfusion control and propagation of intercellular calcium waves (4 -6). Recently pannexin 1 was also shown to form the large pore of the P2X7 purinergic receptor (7, 8). P2X7 plays a major role in inflammation, and its activation by extracellular ATP results in release of interleukin (IL) 2 -1␤ from macrophages, probably involving pannexin 1 as a signaling molecule (7).IL-1␤ production and maturation are tightly regulated by caspase-1 incorporated into large protein complexes termed inflammasomes (9 -11). The molecular composition of the inflammasome depends on the identity of the NOD-like receptor (NLR) family member serving as a scaffold protein in the complex (12). The members of the cytosolic NLR family appear to recognize conserved microbial and viral components termed pathogen-associated molecular patterns in intracellular compartments (13). The bipartite adaptor protein apoptosis-associated speck-like protein containing a CARD (ASC) bridges the interaction between NLR proteins and inflammatory caspases and plays a central role in the assembly of inflammasomes and the activation of caspase-1 in response to a broad range of pathogen-associated molecular patterns and intracellular pathogens (14). In addition, the inflammasome can be activated by danger-associated molecular patterns, molecules endogenous to the organism that signal stress or injury, including extracellular ATP acting at ionotropic P2X7 receptors, fibronectin, or monosodium urate crystals (15,16). Moreover it has been suggested that a rapid K ϩ efflux through ATP-activated P2X7 receptors induces inflammasome assembly (17)(18)(19)(20).Despite the recent advances in the understanding of a...

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“…The cortex was dissected, mechanically dissociated and plated on polyethyleneimine (1 mg/mL) coated culture wells, in Eagle's basal medium (Eurobio, Les Ulis, France), supplemented with 5% horse serum (HS; Eurobio) and 2.5% fetal calf serum (FCS; Eurobio), at a density of 7 Â 10 5 cells/cm 2 . After 2 days in culture the media was replaced with N5 medium [26] with 180 mg/L glucose and changed daily; FCS content was reduced to 1%; Ara-C (cytosine arabinoside, 3 AM, Sigma, Saint Louis, MO, USA) was added to prevent astrocyte proliferation (our cultures were at least 95% neuronal after this treatment), and MK-801 (1 AM, Research Biochemicals International, Natick, MA, USA) to prevent excitotoxicity [7,28].…”

Section: Primary Cultures Of Mouse Cortexmentioning

confidence: 99%

IGF-I protects cortical neurons against ceramide-induced apoptosis via activation of the PI-3K/Akt and ERK pathways; is this protection independent of CREB and Bcl-2?

Willaime‐Morawek

Arbez

Mariani

et al. 2005

Molecular Brain Research

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Current understanding of IGF-I-mediated neuroprotection implies the activation of phosphatidylinositol-3-kinase (PI-3K), which leads to the activation of Akt/Protein Kinase B. In non-neuronal cells, Akt phosphorylates and activates the transcription factor CREB, implicated in the transcription of the anti-apoptotic bcl-2 gene. This paper further analyses the anti-apoptotic IGF-I action in neurons. We show that IGF-I protects cortical neurons against ceramide-induced apoptosis. Ceramide decreases Akt phosphorylation during apoptotic process whereas a simultaneous treatment with IGF-I increases Akt phosphorylation. Analysis of the signal transduction pathways revealed that IGF-I induces CREB phosphorylation via PI-3K and ERK, whereas simultaneous ceramide and IGF-I treatment decreases CREB phosphorylation. Although an overexpression of Bcl-2 protects cortical neurons against ceramide-induced apoptosis, our data indicate that the Bcl-2 protein level is not modulated during IGF-I, ceramide and/or LY294002 treatment. In consequence, we demonstrated that IGF protects neurons against ceramide-induced apoptosis and that IGF-I protection involves the PI-3K/Akt and ERK pathways; this protection may be independent of CREB and Bcl-2. D

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Cryopreservation of primary neurons for tissue culture

Cited by 158 publications

References 8 publications

Pyroptotic Neuronal Cell Death Mediated by the AIM2 Inflammasome

Pyroptotic Neuronal Cell Death Mediated by the AIM2 Inflammasome

The Pannexin 1 Channel Activates the Inflammasome in Neurons and Astrocytes

IGF-I protects cortical neurons against ceramide-induced apoptosis via activation of the PI-3K/Akt and ERK pathways; is this protection independent of CREB and Bcl-2?

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