Techniques for Mammalian Cell Tissue Culture

Phelan, Mary C.

doi:10.1002/0471142727.nsa03bs38

Cited by 47 publications

(108 citation statements)

References 3 publications

(1 reference statement)

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“…c. 24 and 72 hr after infection, replace medium with fresh growth medium. d. 5 days after infection, detach cells with trypsin-EDTA (see Current Protocols article: Phelan, 2007) and fix in 1% formalin. e. Use flow cytometry to determine the % fluorescent cells.…”

Section: Retro Lentiviral Titer and Validation: Flow Cytometry Methodmentioning

confidence: 99%

Production of Viral Constructs for Neuroanatomy, Calcium Imaging, and Optogenetics

et al. 2019

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Advances in design and use of light‐sensitive and light‐emitting sensors have facilitated observation, measurement, and control of neuronal activities. Viruses are effective vectors for delivery of these valuable research tools to mammalian brains. Recombinant viruses are optimized to mediate regulatable, long‐term, and cell‐specific gene expression. Here, we describe production methods for three of the most commonly used types of recombinant viruses in neurobiology research: adeno‐associated virus (AAV), retrovirus/lentivirus, and glycoprotein‐deleted rabies virus. These viral constructs are frequently used for calcium imaging or to deliver neural tracers and optogenetic tools. Popular constructs are readily obtained commercially; however, customized virus production through commercial sources is time consuming and costly. This article aims to provide readers with detailed technical information for rapid production and validation of high‐quality viral particles in a laboratory setting while highlighting advantages and limitations of each viral type. © 2019 by John Wiley & Sons, Inc.

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Section: Retro Lentiviral Titer and Validation: Flow Cytometry Methodmentioning

confidence: 99%

Production of Viral Constructs for Neuroanatomy, Calcium Imaging, and Optogenetics

et al. 2019

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“…Additional reagents and equipment for inhalation anesthesia of rats (Basic Protocol 1), harvesting of spleen and lymph nodes (Reeves & Reeves, 2001), testing cell viability by trypan blue exclusion (APPENDIX 3B; Phelan, 2007), and injection of rodents (Donovan & Brown, 2006) The number of immunized rats depends on the final number of T cells to be transferred per rat. It is common to transfer up to 5 × 10 6 to 2 × 10 7 cells from early cell lines per rat intraperitoneally.…”

Section: Methodsmentioning

confidence: 99%

Lewis Rat Model of Experimental Autoimmune Encephalomyelitis

2017

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In this unit, we describe in detail the most common methods used to break immunological tolerance for central myelin antigens and induce experimental autoimmune encephalomyelitis (EAE) in Lewis rats as an animal model of multiple sclerosis. The resulting disease course ranges from an acute monophasic disease to a chronic relapsing or chronic progressive course, which strongly resembles the human disease. These models enable the study of cellular and humoral autoimmunity against major antigenic epitopes of the myelin basic protein, myelin oligodendrocyte glycoprotein, or proteolipid protein. We provide an overview of common immunization protocols for induction of active and passive EAE, assessment and analysis of clinical score, preparation and purification of myelin basic protein, and derivation of neuroantigen-specific rat T cell lines. Finally, we describe the major clinical characteristics of these models. © 2017 by John Wiley & Sons, Inc.

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“…Wash the NSC culture once in HBSS. (Phelan, 2007) and graft them onto cell culture as described above.…”

Section: Methodsmentioning

confidence: 99%

“…Additional reagents and equipment for trypsinization of cells (Phelan, 2007) 3. Culture NSCs in stem cell medium in a humidified 37°C, 5% CO 2 incubator.…”

Section: % Ethanolmentioning

confidence: 99%

Functional Stem Cell Integration into Neural Networks Assessed by Organotypic Slice Cultures

Forsberg

Thonabulsombat

Jäderstad

et al. 2017

CP Stem Cell Biology

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Re-formation or preservation of functional, electrically active neural networks has been proffered as one of the goals of stem cell-mediated neural therapeutics. A primary issue for a cell therapy approach is the formation of functional contacts between the implanted cells and the host tissue. Therefore, it is of fundamental interest to establish protocols that allow us to delineate a detailed time course of grafted stem cell survival, migration, differentiation, integration, and functional interaction with the host. One option for in vitro studies is to examine the integration of exogenous stem cells into an existing active neural network in ex vivo organotypic cultures. Organotypic cultures leave the structural integrity essentially intact while still allowing the microenvironment to be carefully controlled. This allows detailed studies over time of cellular responses and cell-cell interactions, which are not readily performed in vivo. This unit describes procedures for using organotypic slice cultures as ex vivo model systems for studying neural stem cell and embryonic stem cell engraftment and communication with CNS host tissue. © 2017 by John Wiley & Sons, Inc.

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Techniques for Mammalian Cell Tissue Culture

Cited by 47 publications

References 3 publications

Production of Viral Constructs for Neuroanatomy, Calcium Imaging, and Optogenetics

Production of Viral Constructs for Neuroanatomy, Calcium Imaging, and Optogenetics

Lewis Rat Model of Experimental Autoimmune Encephalomyelitis

Functional Stem Cell Integration into Neural Networks Assessed by Organotypic Slice Cultures

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