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Authors: Ethan Lippmann, Hannah Wilson, Emma Neal
Group: Neurodegeneration Method Development Community

Proper citation: Ethan Lippmann, Hannah Wilson, Emma Neal 2020. Protocol for Subculture of Differentiated Blood-Brain Barrier Endothelial Cells onto Plates and Filters. protocols.io https://dx.doi.org/10.17504/protocols.io.8g5hty6 Copy   

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Authors: Marcos Otero-Garcia, Inma Cobos
Group: Neurodegeneration Method Development Community
Summary: - Protocol based on Krishnaswami et al., Nat Protoc. 2016, 3:499-524- Used routinely in our lab for the isolation of single nuclei from fresh frozen postmortem brains from subjects with neurodegenerative disorders and healthy controls for single-nucleus RNA-seq- Works also for isolation of nuclei from fresh frozen mouse brains - Used for isolation of all nuclei or antibody-enriched populations (i.e., NeuN+ nuclei)- We always perform and strongly recommend FACS if used for single-nucleus RNA-seq

Proper citation: Marcos Otero-Garcia, Inma Cobos 2019. Isolation of single nuclei from postmortem fresh frozen human brain and immunostaining for NeuN. protocols.io https://dx.doi.org/10.17504/protocols.io.6tuhenw Copy   

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Authors: Yingchao Xue, Xiping Zhan, Shisheng Sun, Senthilkumar S. Karuppagounder, Shuli Xia, Valina L Dawson, Ted M Dawson, John Laterra, Jianmin Zhang, Mingyao Ying
Group: Neurodegeneration Method Development Community

Proper citation: Yingchao Xue, Xiping Zhan, Shisheng Sun, Senthilkumar S. Karuppagounder, Shuli Xia, Valina L Dawson, Ted M Dawson, John Laterra, Jianmin Zhang, Mingyao Ying 2020. Quantitative Real‐Time Polymerase Chain Reaction (qRT‐PCR). protocols.io https:// Copy   

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Authors: Michael S. Fernandopulle, Ryan Prestil, Christopher Grunseich, Chao Wang, Li Gan, Michael E. Ward
Group: Neurodegeneration Method Development Community
Summary: Live imaging permits visualization of molecular and organellar dynamics within the neuron. While a standard confocal microscope is sufficient for short imaging experiments, extended imaging applications (>1 hr) are best served by a 37 °C live imaging chamber outfitted onto the microscope. CM should also be changed to Hibernate A Low Fluorescence Medium (BrainBits LLC, cat. no. SKU#HAPR) for extended imaging.

Proper citation: Michael S. Fernandopulle, Ryan Prestil, Christopher Grunseich, Chao Wang, Li Gan, Michael E. Ward 2019. LIVE IMAGING OF i3NEURONS (Support Protocol 5) . protocols.io https://dx.doi.org/10.17504/protocols.io.5w7g7hn Copy   

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Authors: Michael S. Fernandopulle, Ryan Prestil, Christopher Grunseich, Chao Wang, Li Gan, Michael E. Ward
Group: Neurodegeneration Method Development Community
Summary: Following Day 3 replating, i3Neurons should be cultured for at least 1 week before lysis, fixation, or other experimental endpoint, and they remain viable for at least 1 month with proper maintenance. This protocol covers medium conditions, coating of tissue culture dishes with synthetic polymers, recommended plating densities, and maintenance procedures for long-term culture and for specific experimental applications.Cortical Neuron Culture Medium (CM) is sufficient to promote the maturation and longterm maintenance of i3Neurons in culture. i3Neurons express general markers of cortical neurons, as well as specific pre- and postsynaptic markers of glutamatergic excitatory cortical neurons. Since these cells are post-mitotic after 3 days of differentiation (see Basic Protocol 5) and prefer neuron-conditioned medium to fresh medium, maintenance conditions for these cultures are minimal. Generally, half-medium changes every 7 days with fresh, pre-warmed CM are sufficient for culturing beyond d10 (7 days after replating). Neuron attachment and growth also require a strongly adhesive substrate. Coating plates with synthetic polymers such as poly-L-ornithine (PLO) is necessary for i3Neuron attachment, viability, and successful outgrowth.

Proper citation: Michael S. Fernandopulle, Ryan Prestil, Christopher Grunseich, Chao Wang, Li Gan, Michael E. Ward 2019. CULTURING i3NEURONS (Basic Protocol 6). protocols.io https://dx.doi.org/10.17504/protocols.io.5jpg4mn Copy   

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Authors: Andrea Argouarch
Group: Neurodegeneration Method Development Community
Summary: Protocols included in this collection describe cell isolation from human dura mater, culturing, and banking.

Proper citation: Andrea Argouarch 2020. Dural Cell Isolation and Culturing - Collection. protocols.io https://dx.doi.org/10.17504/protocols.io.8m2hu8e Copy   

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Authors: Yingchao Xue, Xiping Zhan, Shisheng Sun, Senthilkumar S. Karuppagounder, Shuli Xia, Valina L Dawson, Ted M Dawson, John Laterra, Jianmin Zhang, Mingyao Ying
Group: Neurodegeneration Method Development Community

Proper citation: Yingchao Xue, Xiping Zhan, Shisheng Sun, Senthilkumar S. Karuppagounder, Shuli Xia, Valina L Dawson, Ted M Dawson, John Laterra, Jianmin Zhang, Mingyao Ying 2020. Protocols for Synthetic mRNAs Drive Highly Efficient iPS Cell Differentiation to Dopaminergic Neurons. protocols.io https:// Copy   

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Authors: Celeste Karch, Rita Martinez, Jacob Marsh
Group: Neurodegeneration Method Development Community

Proper citation: Celeste Karch, Rita Martinez, Jacob Marsh 2019. Serial Dilution of Nucleofected iPSC Pools. protocols.io https://dx.doi.org/10.17504/protocols.io.x76frre Copy   

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Authors: Jennifer B Treweek, Ken Y Chan, Nicholas C Flytzanis, Bin Yang, Benjamin E Deverman, Alon Greenbaum, Antti Lignell, Cheng Xiao, Long Cai, Mark S Ladinsky, Pamela J Bjorkman, Charless C Fowlkes, Viviana Gradinaru
Group: Optical Clearing of Tissue, Neurodegeneration Method Development Community
Summary: To facilitate fine-scale phenotyping of whole specimens, we describe here a set of tissue fixation-embedding, detergent-clearing and staining protocols that can be used to transform excised organs and whole organisms into optically transparent samples within 1–2 weeks without compromising their cellular architecture or endogenous fluorescence. PACT (passive CLARITY technique) and PARS (perfusion-assisted agent release in situ) use tissue-hydrogel hybrids to stabilize tissue biomolecules during selective lipid extraction, resulting in enhanced clearing efficiency and sample integrity. Furthermore, the macromolecule permeability of PACT- and PARS-processed tissue hybrids supports the diffusion of immunolabels throughout intact tissue, whereas RIMS (refractive index matching solution) grants high-resolution imaging at depth by further reducing light scattering in cleared and uncleared samples alike. These methods are adaptable to difficult-to-image tissues, such as bone (PACT-deCAL), and to magnified single-cell visualization (ePACT). Together, these protocols and solutions enable phenotyping of subcellular components and tracing cellular connectivity in intact biological networks.

Proper citation: Jennifer B Treweek, Ken Y Chan, Nicholas C Flytzanis, Bin Yang, Benjamin E Deverman, Alon Greenbaum, Antti Lignell, Cheng Xiao, Long Cai, Mark S Ladinsky, Pamela J Bjorkman, Charless C Fowlkes, Viviana Gradinaru 2019. Whole-body tissue stabilization and selective extractions via tissue-hydrogel hybrids for high-resolution intact circuit mapping and phenotyping. protocols.io https://dx.doi.org/10.17504/protocols.io.8xehxje Copy   

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Authors: Michael S. Fernandopulle, Ryan Prestil, Christopher Grunseich, Chao Wang, Li Gan, Michael E. Ward
Group: Neurodegeneration Method Development Community

Proper citation: Michael S. Fernandopulle, Ryan Prestil, Christopher Grunseich, Chao Wang, Li Gan, Michael E. Ward 2019. ASSESSING RTTA ACTIVITY (Support Protocol 6) . protocols.io https://dx.doi.org/10.17504/protocols.io.5w8g7hw Copy   

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Authors: Ruilin Tian, Jason Hong, Sydney Sattler, Martin Kampmann
Group: Neurodegeneration Method Development Community, KampmannLab

Proper citation: Ruilin Tian, Jason Hong, Sydney Sattler, Martin Kampmann 2019. Lentiviral transduction of iPSCs with sgRNAs and sgRNA libraries. protocols.io https://dx.doi.org/10.17504/protocols.io.8dfhs3n Copy   

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Authors: Yingchao Xue, Xiping Zhan, Shisheng Sun, Senthilkumar S. Karuppagounder, Shuli Xia, Valina L Dawson, Ted M Dawson, John Laterra, Jianmin Zhang, Mingyao Ying
Group: Neurodegeneration Method Development Community

Proper citation: Yingchao Xue, Xiping Zhan, Shisheng Sun, Senthilkumar S. Karuppagounder, Shuli Xia, Valina L Dawson, Ted M Dawson, John Laterra, Jianmin Zhang, Mingyao Ying 2020. Immunofluorescence and Cell Counting. protocols.io https:// Copy   

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Authors: Jeffrey R. Moffitt, Xiaowei Zhuang
Group: Human Cell Atlas Method Development Community, Neurodegeneration Method Development Community
Summary: Quantitative measurements of both the copy number and spatial distribution of large fractions of the transcriptome in single-cells could revolutionize our understanding of a variety of cellular and tissue behaviors in both healthy and diseased states. Single-molecule Fluorescence In Situ Hybridization (smFISH)—an approach where individual RNAs are labeled with fluorescent probes and imaged in their native cellular and tissue context—provides both the copy number and spatial context of RNAs but has been limited in the number of RNA species that can be measured simultaneously. Here we describe Multiplexed Error Robust Fluorescence In Situ Hybridization (MERFISH), a massively parallelized form of smFISH that can image and identify hundreds to thousands of different RNA species simultaneously with high accuracy in individual cells in their native spatial context. We provide detailed protocols on all aspects of MERFISH, including probe design, data collection, and data analysis to allow interested laboratories to perform MERFISH measurements themselves.smFISH is a powerful technique because it allows the quantitative measurement of the exact copy number and spatial distribution of individual mRNAs within single cells in intact tissues. MERFISH extends these powerful abilities to the transcriptome scale by massively multiplexing smFISH measurements by encoding individual RNA species with error-robust barcodes and reading out the barcodes via a series of smFISH measurements. We anticipate this method to be relatively easily adopted by other laboratories and the techniques described in this collection should facilitate these efforts as will the supporting protocols and code that can be found at: http://zhuang.harvard.edu/merfish/.

Proper citation: Jeffrey R. Moffitt, Xiaowei Zhuang 2018. RNA Imaging with Multiplexed Error Robust Fluorescence in situ Hybridization. protocols.io https://dx.doi.org/10.17504/protocols.io.mepc3dn Copy   

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Authors: Celeste Karch, Rita Martinez, Jacob Marsh
Group: Neurodegeneration Method Development Community

Proper citation: Celeste Karch, Rita Martinez, Jacob Marsh 2019. Genomic Editing: iPSC. protocols.io https://dx.doi.org/10.17504/protocols.io.yayfsfw Copy   

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Authors: Michael S. Fernandopulle, Ryan Prestil, Christopher Grunseich, Chao Wang, Li Gan, Michael E. Ward
Group: Neurodegeneration Method Development Community
Summary: Human iPSCs are an ideal system for studying human biology due to their rapid proliferation, genomic stability, and ability to differentiate into many somatic cell types. Historically, specialized culture practices and costly reagents have hindered widespread adoption of iPSCs by the cell biology community. In recent years, however, development of new culture techniques and improved media formulations have dramatically simplified iPSC culture and reduced costs.The protocols described in this unit are adapted from a collection of publications that establish optimal practices for the maintenance of human iPSC cultures (Beers et al., 2012; Ludwig et al., 2006; Chen et al., 2011). While these publications provide useful guidelines for the stem cell novice, here we distill the fundamental procedures necessary for maintaining iPSCs in a pluripotent state and highlight critical steps that may need to be optimized for individual applications. In practice, iPSC lines of interest are usually maintained in an undifferentiated state in small cultures (1 to 3 wells of a 6-well plate) to reduce reagent use before being expanded as needed for experimentation.Essential 8 (E8) is the simplest defined medium for hiPSC culture. E8 may be prepared from its components by the consumer (Table 1; Chen et al., 2011) or purchased as a preformulated kit. Other commercially available media may be substituted, such as mTeSR1 or StemFlex. A volume of 12 ml of medium should be added to each 10-cm tissue culture dish or distributed evenly across each standard multiwell plate (i.e., 2 ml/well for a 6-well plate). E8 should be aspirated and replaced with fresh medium daily, although a double volume may be added at low confluency to permit an extra day of culture without medium changes. StemFlex and E8 Flex contain components that stabilize the recombinant growth factors present in the medium, permitting medium exchange every other day as a general practice. Some iPSC lines (e.g., WTC11) tolerate every-other-day medium changes of standard E8 medium without loss of pluripotency or cell death, further reducing costs of medium and consumables. Finally, mTeSR1 may promote cell survival in stressful conditions better than E8, especially for finicky iPSC lines, although supplementation with a ROCK inhibitor (RI) is also recommended in such scenarios. Use of standard E8 will be assumed throughout this basic protocol.Additionally, this protocol uses Matrigel-coated tissue culture plates. Matrigel works well for iPSC culture and has been widely adopted for research applications. However, since Matrigel is derived from murine sarcoma cells, it is not chemically defined and exhibits batch-to-batch variability. Alternative defined coatings include recombinant laminin or vitronectin, although these substrates are typically more costly. Notably, downstream neural differentiation described in these protocols occurs in fully defined conditions (see Basic Protocols 5 to 8), so the choice of iPSC substrate is of minimal scientific importance to all but clinical-grade applications.

Proper citation: Michael S. Fernandopulle, Ryan Prestil, Christopher Grunseich, Chao Wang, Li Gan, Michael E. Ward 2019. MAINTENANCE CULTURE OF iPSCs (Basic Protocol 1). protocols.io https://dx.doi.org/10.17504/protocols.io.48vgzw6 Copy   

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Authors: Connor Ludwig, Ruilin Tian, Martin Kampmann
Group: Neurodegeneration Method Development Community, KampmannLab
Summary: This protocol explains general culture and maintenance of the WTc11 iPSC cell line.

Proper citation: Connor Ludwig, Ruilin Tian, Martin Kampmann 2019. WTc11 iPSC Culture and Maintenance. protocols.io https://dx.doi.org/10.17504/protocols.io.8rhhv36 Copy   

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Authors: Sejin Yoon, Sergiu P. Pașca
Group: Neurodegeneration Method Development Community

Proper citation: Sejin Yoon, Sergiu P. Pașca 2020. Generation of hCS from hiPSC maintained in feeder-free conditions. protocols.io https:// Copy   

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Authors: Andrea Argouarch
Group: Neurodegeneration Method Development Community
Summary: Protocol includes banking dural cells into cryovials for long term storage in liquid nitrogen.

Proper citation: Andrea Argouarch 2020. Freezing/Cell Banking. protocols.io https://dx.doi.org/10.17504/protocols.io.8gmhtu6 Copy   

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Authors: Steven A. Sloan, Jimena Anderson, Anca M. Pașca, Fikri Birey, Sergiu Pasca
Group: Neurodegeneration Method Development Community

Proper citation: Steven A. Sloan, Jimena Anderson, Anca M. Pașca, Fikri Birey, Sergiu Pasca 2020. Generation and assembly of human brain region-specific organoids. protocols.io https:// Copy   

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Authors: Schuele Lab, Faria Zafar
Group: Neurodegeneration Method Development Community, Schuele lab_1
Summary: The purpose of this protocol is high-molecular weight DNA extraction from whole blood for genetic analyses, including no amplification long-range sequencing.

Proper citation: Schuele Lab, Faria Zafar 2020. DNA extraction from whole blood using simple salting out procedure. protocols.io https://dx.doi.org/10.17504/protocols.io.bbzqip5w Copy   

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