Searching the RRID Resource Information Network
Are you sure you want to leave this community? Leaving the community will revoke any permissions you have been granted in this community.
Authors: Celeste Karch, Rita Martinez, Jacob Marsh
Group: Neurodegeneration Method Development Community
Proper citation: Celeste Karch, Rita Martinez, Jacob Marsh 2019. Splitting 96 Well Plates for gDNA Extraction and Continuing Culture. protocols.io dx.doi.org/10.17504/protocols.io.x78frrw Copy
Authors: Andrea Argouarch
Group: Neurodegeneration Method Development Community
Summary: Protocol includes feeding of T75 flask, T150 flask, or the back up 6 well plate.
Proper citation: Andrea Argouarch 2020. Feeding T75, T150, and 6wp Backup. protocols.io dx.doi.org/10.17504/protocols.io.8gkhtuw Copy
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 dx.doi.org/10.17504/protocols.io.x62frge Copy
Authors: Celeste Karch, Rita Martinez, Jacob Marsh
Group: Neurodegeneration Method Development Community
Proper citation: Celeste Karch, Rita Martinez, Jacob Marsh 2019. Neural progenitor banking. protocols.io dx.doi.org/10.17504/protocols.io.x89frz6 Copy
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 lipid-mediated transfection of iPSCs maintained in E8 medium on Matrigel. While several lipid-based transfection reagents are commercially available, Lipofectamine Stem is used here because it is specifically optimized for delivery of DNA plasmids into hiPSCs. If preferred, similar results may also be achieved by electroporation or nucleofection, and other lipid reagents are available for in vitro-transcribed RNA or in vitro-translated ribonucleoproteins (RNPs). Furthermore, while this protocol provides the steps for a general transfection, specific details are provided below regarding insertion of the transgene cassettes relevant for neural differentiation (See Basic Protocols 5 and 7).
Proper citation: Michael S. Fernandopulle, Ryan Prestil, Christopher Grunseich, Chao Wang, Li Gan, Michael E. Ward 2019. LIPID-MEDIATED TRANSFECTION OF iPSCs (Basic Protocol 2). protocols.io dx.doi.org/10.17504/protocols.io.5deg23e Copy
Authors: Ethan Lippmann, Hannah Wilson, Emma Neal
Group: Neurodegeneration Method Development Community
Summary: Standardized single cell seeding protocol for Blood-Brain Barrier (BBB) differentiation.
Proper citation: Ethan Lippmann, Hannah Wilson, Emma Neal 2020. Single Cell Seeding of BBB Stem Cell Model. protocols.io dx.doi.org/10.17504/protocols.io.8j9hur6 Copy
Authors: Celeste Karch, Rita Martinez, Jacob Marsh
Group: Neurodegeneration Method Development Community
Proper citation: Celeste Karch, Rita Martinez, Jacob Marsh 2019. Splitting 96 Well Plates for gDNA Extraction and Freezing Down. protocols.io dx.doi.org/10.17504/protocols.io.x79frr6 Copy
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 dx.doi.org/10.17504/protocols.io.8rhhv36 Copy
Authors: Michael S. Fernandopulle, Ryan Prestil, Christopher Grunseich, Chao Wang, Li Gan, Michael E. Ward
Group: Neurodegeneration Method Development Community
Summary: This protocol will use genomic DNA isolated from the purified iPSC clones with CLYBL or AAVS1 gene insertion to determine if integration of the transgene has occurred correctly, in a heterozygous or homozygous fashion, and if the floxed selection genes are present. Primer sequences, amplicon sizes, PCR mix composition, and thermal conditions are included in the steps.
Proper citation: Michael S. Fernandopulle, Ryan Prestil, Christopher Grunseich, Chao Wang, Li Gan, Michael E. Ward 2019. GENOTYPING OF iPSCS WITH GENE INSERTIONS (Support Protocol 1). protocols.io dx.doi.org/10.17504/protocols.io.5u9g6z6 Copy
Authors: Merissa Chen, Nina Draeger, Martin Kampmann, Kun Leng, Emmy Li, Connor Ludwig, Gregory Mohl, Avi Samelson, Sydney Sattler, Ruilin Tian
Group: Neurodegeneration Method Development Community, KampmannLab
Summary: This protocol describes thedifferentiation of iPSCs with stably integrated doxycycline-inducible Ngn2 (such as i3Ns).
Proper citation: Merissa Chen, Nina Draeger, Martin Kampmann, Kun Leng, Emmy Li, Connor Ludwig, Gregory Mohl, Avi Samelson, Sydney Sattler, Ruilin Tian 2019. iNeuron pre-differentiation & differentiation protocol. protocols.io dx.doi.org/10.17504/protocols.io.8dths6n Copy
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 dx.doi.org/10.17504/protocols.io.5tng6me Copy
Authors: Jacob Marsh, Celeste Karch
Group: Neurodegeneration Method Development Community
Summary: This protocol is about Maintenance and Expansion of induced pluripotent stem cells.
Proper citation: Jacob Marsh, Celeste Karch 2020. iPSC Cell Culture – Maintenance and Expansion. protocols.io dx.doi.org/10.17504/protocols.io.9pwh5pe Copy
Authors: Andrea Argouarch
Group: Neurodegeneration Method Development Community
Summary: Protocol includes splitting growing cell line from a T75 flask into two T150 flasks for expansion.
Proper citation: Andrea Argouarch 2020. Splitting p1 (1xT75) to p2 (2xT150). protocols.io dx.doi.org/10.17504/protocols.io.8gnhtve Copy
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 dx.doi.org/10.17504/protocols.io.be43jgyn Copy
Authors: Cortina Chen, Iman Mali, Florian T Merkle
Group: Neurodegeneration Method Development Community
Summary: This protocol is about Differentiation of hPSCs to hypothalamic neurons.
Proper citation: Cortina Chen, Iman Mali, Florian T Merkle 2020. Differentiation of hPSCs to hypothalamic neurons. protocols.io dx.doi.org/10.17504/protocols.io.bn8qmhvw Copy
Authors: Michael S. Fernandopulle, Ryan Prestil, Christopher Grunseich, Chao Wang, Li Gan, Michael E. Ward
Group: Neurodegeneration Method Development Community
Summary: While lipid-mediated transient transfection and expression of transgenes in hiPSCs is often quite high (> 50 %), even an optimal transfection with the best editing tools can result in very low genomic integration efficiency (Basic Protocol 4).In order to remove non-edited cells, positive selection markers such as genes coding for fluorescent proteins or antibiotic resistance are often included in the insert construct under constitutive promoters, and this protocol describes general methods for the former strategy. However, constant expression of a fluorophore interferes with immunocytochemistry, and antibiotic selection can incorrectly select for cells with multiple aberrant transgene insertions. To counter these shortcomings, we developed a platform for enrichment from heterogeneous cell populations through cell-surface affinity for magnetic streptavidin beads mediated by expression of a streptavidin binding peptide (SBP) fused to the truncated extracellular and transmembrane domains of low-affinity nerve growth factor receptor (LNGFR). The SBP tag is commonly used to facilitate co-immunoprecipitation with streptavidin-coated beads, and, with fusion to the LNGFR, it is efficiently localized to the extracellular surface where it can bind the beads more readily.This construct was developed based on one initially designed for purification of transgenic T cells in suspension (Matheson, Peden, & Lehner, 2014), and it is included in Addgene cat. no. 105842 for direct use with hNIL or excision and insertion into other constructs.Here, we extend the original protocol in order to optimize enrichment following safe harbor locus insertion in iPSCs, and we present evidence to support its use as either a positive or negative selection marker in a variety of transgenic applications. However, we have found that the SBP-LNGFR construct should be the only gene expressed under a dedicated, highly active promoter, as it appears to interfere with 2A-mediated ribosome skipping. For example, a different construct, with the reverse tetracycline transactivator (rtTA) linked to SBP-LNGFR by a T2A sequence, led to disrupted differentiation due to reduced rtTA activity (see Support Protocol 6).When structured correctly, however, SBP-LNGFR permits highly selective enrichment that does not require specialized equipment as in fluorescence-activated cell sorting (FACS) and does not require extensive tuning as in antibiotic selection. Notably, the efficiency of enrichment increases exponentially with a lower frequency of positive cells in the population. In addition, the protocol is easily modified to permit negative selection, e.g., for removing a floxed transgene with Cre recombinase or a gene knockout reporter. Furthermore, cells expressing SBP-LNGFR may be transiently labeled at the cell surface by incubating with any fluorophore conjugated to streptavidin. After washing to remove unbound fluorophore, these cells may be imaged, marked for clonal picking, or FACS-purified at an equivalent efficacy to cells constitutively expressing a fluorophore marker of insertion. Fluorescence then returns to undetectable levels within one passage and does not interfere with downstream immunocytochemistry applications.Precise, label-free editing of endogenous loci may be enriched by FACS sorting for transient expression of the components necessary for editing 1 day after transfection [i.e., using a combined gRNA, Cas9, and GFP expression plasmid with an integration plasmid expressing RFP outside of the insertion sequence or by using functionalized RNPs such as S1mplex with Qdots or fluorescent streptavidin (Carlson-Stevermer et al., 2017)]. This can considerably increase the prevalence of correctly edited cells, but the combined stress of transfection and sorting can decrease cell survival. To compensate, it is recommended to transfect and sort (2.4 × 106 cells) in triplicate and to plate cells at high density following the sort. In addition, extensive downstream cloning and genotyping is often required due to the propensity for non-homologous end joining leading to mutagenesis at the target locus rather than homology-directed repair-mediated integration of the desired insert.This protocol will continue with the assumption of integration of a transgene cassette into a safe-harbor locus as described in Basic Protocol 2. Label-free gene knockouts caused by NHEJ-mediated indel formation are typically prevalent (> 15 %), so frontline enrichment is optional for these applications. Following enrichment, small stocks of heterogeneous cell populations should be frozen, and the cells should be taken directly to Basic Protocol 4 for clonal isolation.
Proper citation: Michael S. Fernandopulle, Ryan Prestil, Christopher Grunseich, Chao Wang, Li Gan, Michael E. Ward 2019. SELECTION AND ENRICHMENT OF TRANSGENIC CELL POPULATIONS (Basic Protocol 3). protocols.io dx.doi.org/10.17504/protocols.io.5fvg3n6 Copy
Authors: Celeste Karch, Rita Martinez, Jacob Marsh
Group: Neurodegeneration Method Development Community
Proper citation: Celeste Karch, Rita Martinez, Jacob Marsh 2019. iPSC Freezing. protocols.io dx.doi.org/10.17504/protocols.io.x8zfrx6 Copy
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 dx.doi.org/10.17504/protocols.io.bkrvkv66 Copy
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 dx.doi.org/10.17504/protocols.io.5u5g6y6 Copy
Authors: Vijay Singh, Marta Castellana-Cruz, Nunilo Cremades, Laura Volpicelli-Daley
Group: Neurodegeneration Method Development Community
Summary: Animal models that accurately recapitulate the accumulation of alpha-synuclein (α-syn) inclusions, progressive neurodegeneration of the nigrostriatal system and motor deficits can be useful tools for Parkinson's disease (PD) research. The preformed fibril (PFF) synucleinopathy model in rodents generally displays these PD-relevant features, however, the magnitude and predictability of these events is far from established. We therefore have optimized the synthesis generation of α-syn fibrils to ensure reliable, robust results. These fibrils can be added to neurons in culture, differentiated iPSCs, or injected into mice or rats. The protocol includes steps for fibril synthesis as well as sonication for fibril fragmentaion which is a critical step for inducing formation of α-syn inclusions.
Proper citation: Vijay Singh, Marta Castellana-Cruz, Nunilo Cremades, Laura Volpicelli-Daley 2020. Generation and Sonication of α-synuclein Fibrils. protocols.io dx.doi.org/10.17504/protocols.io.bhhrj356 Copy
Can't find your Protocol?
We recommend that you click next to the search bar to check some helpful tips on searches and refine your search firstly. If you want to find a specific protocol and you know the DOI of the protocol already, it's easier to enter a DOI to search. You can refine the search results using Facets on the left side of the search results page. If you are on the table view, you can also search in a specific column by clicking the column title and enter the keywords.
If you still could not find your protocol in the search results, please help us by adding it into the system — it's easy. Create and publish your protocols at Protocols.io.
Welcome to the dkNET Resources search. From here you can search through a compilation of resources used by dkNET and see how data is organized within our community.
You are currently on the Community Resources tab looking through categories and sources that dkNET has compiled. You can navigate through those categories from here or change to a different tab to execute your search through. Each tab gives a different perspective on data.
If you have an account on dkNET then you can log in from here to get additional features in dkNET such as Collections, Saved Searches, and managing Resources.
Here is the search term that is being executed, you can type in anything you want to search for. Some tips to help searching:
You can save any searches you perform for quick access to later from here.
We recognized your search term and included synonyms and inferred terms along side your term to help get the data you are looking for.
If you are logged into dkNET you can add data records to your collections to create custom spreadsheets across multiple sources of data.
Here are the sources that were queried against in your search that you can investigate further.
Here are the categories present within dkNET that you can filter your data on
Here are the subcategories present within this category that you can filter your data on
If you have any further questions please check out our FAQs Page to ask questions and see our tutorials. Click this button to view this tutorial again.
The NIDDK Information Network (dkNET) serves the needs of basic and clinical investigators by providing seamless access to large pools of data and research resources relevant to the mission of The National Institute of Diabetes Digestive and Kidney Diseases (NIDDK). dkNET is hosted at University of California San Diego, and is supported by NIH NIDDK grant U24DK097771.
Email: info_at_ dknet.org
