Midbrain organoid differentiation in spinner flasks
By onMidbrain differentiation protocol using spinner flasks
Nucleofection (Amaxa) and electroporation (Biorad) of hPSCs
By onThis collection describes the standard procedure for the delivery of plasmids, mRNA or ribonucleoprotein (RNP) into human pluripotent stem cells (hPSCs) using nucleofection or electroporation.
Electroporation of hPSCs
By onThis protocol describes the standard procedure for the delivery of plasmids into human pluripotent stem cells (hPSCs) using electroporation.
Differentiation of human cortical neurons (CNs) from induced pluripotent stem cells (iPSCs) and their coculture with rat astrocytes
By onThis protocol described the production of human cortical neurons from induced pluripotent stem cells in cocultured with commercially available rat astrocytes.
Generation of knock-in and knock-out CRISPR/Cas9 editing in mammalian cells
By onThis protocol is to help with generation of knock-in and knock-out CRISPR/Cas9 editing in mammalian cells
Generation of induced pluripotent stem cells and gene correction
By oniPSC generation and gene correction (CRISPR-CAS9) protocol.
Astrocyte isolation and culturing
By onThis protocol details steps for preparing an astrocyte monoculture from mouse pups. From dissection to plated samples ready to be transfected, treated, lysed, or imaged, the protocol takes 20-25 days.
Plasmid-reprogramming of human fibroblasts
By onThis protocol is part of a collection of protocols for the paper, "Glucocerebrosidase, a Parkinson's disease-associated protein, is imported into mitochondria and regulates complex I assembly and function"
Differentiation of human medium spiny neurons (MSNs) from induced pluripotent stem cells (iPSCs)
By onThis protocol generates human medium spiny neurons (MSNs) from induced human pluripotent stem cells (iPSCs).
Seeding nucleofected hPSCs in 96-well plates using limited dilution
By onThis protocol describes a standard procedure for seeding nucleofected human pluripotent stem cells in 96-well plates using limited dilution. This protocol follows nucleofection of hPSCs as described in detail in the collection "Nucleofection (Amaxa) and electroporation (Biorad) of hPSCs;" dx.doi.org/10.17504/protocols.io.b4qnqvve
Generation of stable ATG3-mCherry wild-type or mutans HeLa cells with HaloTag-LC3B using lentivirus
By onThis protocol details the generation of stable ATG3-mCherry wild-type or mutant HeLa cells with HaloTag-LC3B using lentivirus.
Adapting hPSCs cultured on MEFs to feeder-free system
By onThis protocol describes the procedure of adapting human pluripotent stem cells (hPSCs) to feeder-free culturing conditions using mTeSR-plus or StemFlex
Regular maintenance of human pluripotent stem cells
By onThis protocol describes the regular maintenance and passaging human iPSCs.
Cell line construction and maintenance for Lyso-IP and Endo-IP analysis of amyloid precursor protein processing
By onLyso-IP is a method that allows for the isolation of lysosomes for proteomics and metabolomics (dx.doi.org/10.17504/protocols.io.bybjpskn; dx.doi.org/10.17504/protocols.io.bx9hpr36). We have developed an analogous approach for purification of early/sorting endosomes (Endo-IP). In addition, we have found that endolysosomal purification via Lyso-IP and Endo-IP can be coupled with a quantitative proteomics workflow to obtain snapshots of Amyloid Precursor Protein (APP) processing to its Aβ products (Park et al. in submission). Here, we describe methods for cell line construction and maintenance of 293 cells with TMEM192-3xHA and 3xFLAG-EEA1, which are used for lysosome and endosome purification, respectively, with the addition of patient mutations to APP promotes processing. Cells with endogenously tagged TMEM192 and stably expressing FLAG-EEA1 are referred to as 293EL cells, for Endo-IP and Lyso-IP. These cells were also prepared in a form that has a deletion of the APP gene (293EL;APP-/-) and the same cells reconstituted with a lentivirus stably expressing APPSw;T700N to allow functional analysis of APP processing.
Passaging of hPSCs grown on MEFs
By onThis protocol describes the standard procedure of using collagenase to passage human pluripotent stem cells (hPSCs) on inactivated mouse embryonic fibroblasts (MEFs).
Freezing of mouse embryonic fibroblasts (MEFs) for hPSC cultures
By onThis protocol describes the freezing of mouse embryonic fibroblasts (MEFs), which can later be used as feeder cells for human pluripotent stem cell (hPSC) culture.
Piggybac-mediated stable expression of NGN2 in iPSCs for differentiation into excitatory glutamatergic neurons V.2
By onThe authors adapted a previously described method for employing Piggybac transfection to stably express doxycycline-inducible NGN2 in human iPSCs.
Generation of stable cell lines via lentiviral transduction
By onProtocol describing the generation of stable cell lines using a lentivirus system.
Differentiation NPCs to Dopaminergic/Midbrain Neurons
By onThis protocol details methods for differentiation of NPCs to Dopaminergic/Midbrain Neurons. This protocol is part of a Collection of protocols (dx.doi.org/10.17504/protocols.io.8epv593dng1b/v1) for the paper "Glucocerebrosidase, a Parkinson´s disease-associated protein, is imported into mitochondria and regulates complex I assembly and function" (https://doi.org/10.21203/rs.3.rs-1521848/v1)
