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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. Nucleofection of iPSC. protocols.io https://dx.doi.org/10.17504/protocols.io.x62frge 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: PrimersCLYBL insertion of hNIL CLYBL insertion of mag-hNIL

Proper citation: Michael S. Fernandopulle, Ryan Prestil, Christopher Grunseich, Chao Wang, Li Gan, Michael E. Ward 2019. REAGENTS AND SOLUTIONS (Support Protocol 7.2). protocols.io https://dx.doi.org/10.17504/protocols.io.5vag62e 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. Thawing Rosettes and NPC. protocols.io https://dx.doi.org/10.17504/protocols.io.x9afr2e Copy   

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Authors: Marcos Otero-Garcia, Inma Cobos
Group: Neurodegeneration Method Development Community
Summary: - Protocol optimized to isolate single neuronal somas with cytoplasmic protein aggregates from postmortem fresh frozen brain- Works for the isolation of neuronal somas with neurofibrillary tangles from Alzheimer disease's brains- Note that cell membranes are highly disrupted in tissues that were previously frozen. Thus, cytoplasmic transcripts are largely lost. This protocol is therefore useful for the isolation of specific cell populations using antibodies against cytoplasmic antigens, but not for the profiling of cytoplasmic mRNA.

Proper citation: Marcos Otero-Garcia, Inma Cobos 2019. Isolation of single somas from postmortem fresh frozen human brain and immunostaining for AT8 and MAP2. protocols.io https://dx.doi.org/10.17504/protocols.io.6uqhevw 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: Staining i3Neurons for immunofluorescence (IF) studies is difficult due to the delicate and interconnected nature of the neuronal processes. These processes are easily disrupted in the extensive series of washes in an IF study, and initial dissociation of even a few processes often results in entire sheets of neurons lifting off the culture surface. The best ways to minimize these events are by reducing the number of total washes and by carrying out washes slowly on a tilted dish. Additionally, if possible, IF studies should only be done in neurons 10 days old or younger. After this point, neurons tend to be very delicate and even extremely gentle washing typically causes substantial cell washout.

Proper citation: Michael S. Fernandopulle, Ryan Prestil, Christopher Grunseich, Chao Wang, Li Gan, Michael E. Ward 2019. IMMUNOCYTOCHEMISTRY OF i3NEURONS (Support Protocol 2). protocols.io https://dx.doi.org/10.17504/protocols.io.5wtg7en Copy   

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Authors: Andrea Argouarch
Group: Neurodegeneration Method Development Community
Summary: Isolation of cells from human dura mater. Protocol includes tissue freezing, cutting the tissue with surgical tools, plating into a 6 well plate, placing a coverslip on top of the tissue, and adding cell culture media.

Proper citation: Andrea Argouarch 2020. Dural Cell Isolation . protocols.io https://dx.doi.org/10.17504/protocols.io.8ghhtt6 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. Electrophysiological Recordings. 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. Thawing iPSC Plate. protocols.io https://dx.doi.org/10.17504/protocols.io.x8ufrww Copy   

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

Proper citation: Jacob Marsh, Rj Martinez, Celeste Karch 2020. Thawing, Expanding, and Freezing Human Fibroblasts. protocols.io https://dx.doi.org/10.17504/protocols.io.bgunjwve 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: Lentiviral infection of neurons can be used for a variety of applications in microscopy and biochemistry, provided that the transgene is driven by promoters not silenced in iPSCs or neurons (e.g., CAG, PGK In EF-1α, not CMV, see Critical Parameters and Troubleshooting). The precise parameters for optimal viral production and supernatant collection/concentration will depend on the type of virus used, viral packaging mechanism, viral packaging cell line, and specific transgene introduced. Highly efficient results have been observed with lentivirus produced by Lenti-X HEK cells, with transgenes carried in the pLEX backbone.

Proper citation: Michael S. Fernandopulle, Ryan Prestil, Christopher Grunseich, Chao Wang, Li Gan, Michael E. Ward 2019. TRANSDUCTION OF i3NEURONS (Support Protocol 4). protocols.io https://dx.doi.org/10.17504/protocols.io.5wvg7e6 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, Metabolomics Protocols & Workflows

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. Immunoprecipitation (IP) and Mass Spectrometry. protocols.io https:// Copy   

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Authors: Andrea Argouarch
Group: Neurodegeneration Method Development Community
Summary: Detection of mycoplasma utilizing the bulldog-bio kit. Steps include, gDNA isolation from cells, nanodrop, PCR, and running an agarose gel to detect mycoplasma within a cell culture line. Image from https://cellculturedish.com/cell-culture-basics-mycoplasma-101-a-practical-guide-to-prevention-detection-and-elimination-of-mycoplasma-contamination/

Proper citation: Andrea Argouarch 2020. Mycoplasma. protocols.io https://dx.doi.org/10.17504/protocols.io.8gphtvn 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: This protocol describes the rapid and robust differentiation of hiPSCs into lower motor neurons (i3LMNs) via induced expression of the transcription factors NGN2, ISL1, and LHX3 (hNIL) (Mazzoni et al., 2013; Shi et al., 2017). In particular, a donor construct containing these factors under the tetracycline response element (TRE3G) (Gossen & Bujard, 1992), a CAG promoter driving constitutive expression of the reverse tetracycline transactivator (rtTA3G), and an EF-1α promoter driving constitutive expression of selection genes (mCherry for Addgene, cat. no. 105841 and SBP-LNGFR and mApple for Addgene, cat. no. 105842) was stably integrated into the safe-harbor CLYBL locus via TALENs (Cerbini et al., 2015). Motor neuron differentiation efficiency can vary between iPSC lines, and homozygous insertion into both CLYBL alleles may result in improved efficiency. Reduced differentiation efficiency of mCherry positive clones can occur with off-target integration of the donor construct. Additionally, the mCherry reporter is flanked by loxP sites, permitting excision by transient transfection of Cre recombinase if desired.

Proper citation: Michael S. Fernandopulle, Ryan Prestil, Christopher Grunseich, Chao Wang, Li Gan, Michael E. Ward 2019. DIFFERENTIATION OF i3LMNS (Basic Protocol 7). protocols.io https://dx.doi.org/10.17504/protocols.io.5tng6me Copy   

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Authors: Simone Codeluppi, Lars Borm, amit zeisel, Gioele La Manno, Josina A. van Lunteren, Camilla I. Svensson, Sten Linnarsson
Group: Human Cell Atlas Method Development Community, CZI Spatial Transcriptomics Protocol Repository, Neurodegeneration Method Development Community
Summary: osmFISH is a cyclic single molecule fluorescent in situ hybridization protocol used to quantify the expression level of specific transcripts in tissue sections by direct labeling of individual RNA molecules. The number of transcripts quantified in each round correspond the numbers of fluorophores available in the microscope setup. In order to quantify a large number of genes, osmFISH provides a method to remove the probes/labelling (stripping) from their targets and get the tissue ready for the next round of labeling - imaging - stripping.Even though the osmFISH protocol has been developed to perform multiple smFISH rounds, it can also be used as a quick and simple method for one-round smFISH. Furthermore, the protocol can be further extended to include more complex encoding/barcoding shemes that can be used to resolve a larger number of targets. 

Proper citation: Simone Codeluppi, Lars Borm, amit zeisel, Gioele La Manno, Josina A. van Lunteren, Camilla I. Svensson, Sten Linnarsson 2018. osmFISH. protocols.io https://dx.doi.org/10.17504/protocols.io.psednbe Copy   

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Authors: Fabia Filipello, Jacob Marsh, Rj Martinez, Celeste Karch
Group: Neurodegeneration Method Development Community
Summary: This protocol outlines the derivation of Hematopoietic Progenitor Cells and differentiation of iMGLs using iPSC cultures. This protocol is modified the following papers.

Proper citation: Fabia Filipello, Jacob Marsh, Rj Martinez, Celeste Karch 2020. Differentiation of iPSC into Microglia-Like Cells (iMGL). protocols.io https://dx.doi.org/10.17504/protocols.io.be43jgyn 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. 6-Hydroxydopamine (6-OHDA) Treatment and Neurite Tracing in miDA 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. Sanger Sequencing. protocols.io https://dx.doi.org/10.17504/protocols.io.x8tfrwn Copy   

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Authors: Jeffrey R. Moffitt, Xiaowei Zhuang
Group: Human Cell Atlas Method Development Community, Neurodegeneration Method Development Community
Summary: The preparation and staining of samples for MERFISH follows closely the typical protocols used for smFISH (Raj et al., 2008). However, there are a few places in which we have modified these protocols to optimize MERFISH staining. Again, RNase contamination can destroy samples, so care should be taken to work in an RNase-free environment.

Proper citation: Jeffrey R. Moffitt, Xiaowei Zhuang 2018. RNA Imaging with MERFISH - Sample Preparation and Staining. protocols.io https://dx.doi.org/10.17504/protocols.io.merc3d6 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: Induction and differentiation of i3LMNs is nearly identical to the first 3 days of differentiation for i3Neurons (see Basic Protocol 5), including identical induction medium. Following replating, however, differences arise including the use of Motor Neuron Culture Medium (MM) for long-term culture (Table 5), additional reagents to reduce proliferative cells if necessary, and variable options for coating polymers.MM is sufficient to promote the maturation and long-term culture of i3LMNs. While a majority of these cells at Day 3 are committed to differentiation to post-mitotic neurons, a small subset may remain proliferative and can quickly overtake the culture. To compensate, a 1-day pulse of bromodeoxyuridine (BrdU) is recommended at the time of replating of Day 3 i3LMNs and has proven effective at impairing mitosis without causing neural toxicity. Following BrdU treatment, medium should be completely exchanged the following day. CultureOne is also effective at reducing levels of proliferative cells over time, and it may be included in MM medium with usually minimal effects on neural cell health. In general, one fourth to one half of the medium should be aspirated and replaced with fresh medium every 3 to 4 days.Neuron attachment and growth also requires a strongly adhesive substrate. Coating plates with synthetic polymers such as poly-L-ornithine (PLO), polyethyleneimine (PEI), or poly-D-lysine (PDL) is sufficient for cell attachment, and providing an optional additional coating of purified laminin improves i3LMN viability and neurite outgrowth. Laminincoated wells also support proliferative cells better than polymer without laminin, so BrdU is necessary in these conditions.While these substrates are stiffer than those under biological conditions, they reduce cell migration and clumping, facilitating imaging of individual cells. In our experience, PLO has produced the best neuronal morphology, but it is also the most sensitive to cell detachment resulting from medium exchanges. Detachment is a particular concern at high cell density and after extended time in culture, as the interconnected network of neural processes can cause entire wells to detach from the edges. PEI and PDL typically promote stronger adhesion, but PEI is toxic to cells if coating is not performed properly, and rapid degradation and batch-to-batch variability complicate the use of PDL. Cells are especially susceptible to detachment during the many washes required for immunocytochemistry, so these steps should be performed with extreme care. This protocol will assume use of PLO, although coating with PEI or PDL may be performed using an identical protocol except where noted.

Proper citation: Michael S. Fernandopulle, Ryan Prestil, Christopher Grunseich, Chao Wang, Li Gan, Michael E. Ward 2019. CULTURING i3LMNS (Basic Protocol 8). protocols.io https://dx.doi.org/10.17504/protocols.io.5u5g6y6 Copy   

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Authors: Celeste Karch, Rita Martinez, Jacob Marsh
Group: Neurodegeneration Method Development Community
Summary: All methods described in this collection should be performed using sterile technique.

Proper citation: Celeste Karch, Rita Martinez, Jacob Marsh 2019. iPSC Cortical Differentiation. protocols.io https://dx.doi.org/10.17504/protocols.io.x9cfr2w Copy   

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