Cryosectioning mouse brain
By savannah onThis protocol details the cryosectioning of the mouse brain.
Plasmid-reprogramming of human iPSCs
By savannah onThis protocol details about plasmid-reprogramming of human fibroblasts.
Cell line construction and maintenance for Lyso-IP with or without genes linked with lysosomal storage disease
By savannah onAssociated with the following preprint (published September 30th 2021): Progranulin deficiency results in reduced bis(monoacylglycero)phosphate (BMP) levels and gangliosidosis. bioRxiv 2021.09.30.461806; doi: https://doi.org/10.1101/2021.09.30.461806
Endosomal and lysosomal immunoprecipitation for proteomics, lipidomics, and TEM
By savannah onHere, authors describe an approach for purification of early/sorting endosomes, providing a means by which to examine early aspects of the endolysosomal system (Endo-IP) and to combine this with lysosome purification using Lyso-IP. This allows one to examine the proteome, lipidome, as well as electron microscopy imaging of endosomes.
Immunofluorescence of RAB5 and FLAG-EEA1 puncta after Dynamin-1 and -2 inhibition with Dyngo4a
By savannah onHere the authors present an immunofluorescence protocol to assess the extent of colocalization between FLAG-EEA1 and RAB5 with and without the Dynamin-1 and -2 (DNM1/2) inhibitor Dyngo4a.
In vitro GCase activity assay (total cell lysate)
By savannah onAt acid pH B–glucosidase hydrolyses the substrate 4–methylumbelliferyl–B–D–glucopyranoside to 4–methylumbelliferone and glucose. Adding alkaline buffer stops the enzyme reaction and causes 4–methylumbelliferone to fluoresce at a different wavelength from unhydrolyzed substrate, thereby permitting its measurement in the presence of a vast excess of unhydrolyzed substrate.
Lipidomic analysis of tissue culture cells, tissues, and purified organelles
By savannah onThe objective of this protocol is to provide directive on how to extract lipids from plasma, cells, tissue, and purified organelles for analysis by liquid chromatography (LC)-MS. This will typically yield quantitative data for more than 200 lipids, depending on the sample type analyzed, across a range of lipid classes: phospholipids, cardiolipins, sphingolipids, di- and triacyclglycerols, and cholesterylesters.
Passaging of hPSCs grown on MEFs
By savannah onThis protocol describes the standard procedure of using collagenase to passage human pluripotent stem cells (hPSCs) on inactivated mouse embryonic fibroblasts (MEFs).
LRRK2 RCKW protein purification
By savannah onProtein purification protocol for tag-less LRRK2RCKW as done by Leschziner and Reck-Peterson Labs. Same protocol can be used to purify LRRK1RCKW as well. Original protocol by David Snead. Modified by Yu Xuan Lin and Mariusz Matyszewski for publication.
Thawing, passaging, and freezing of hPSCs on MEFs
By savannah onThis collection contains protocols which describe the standard procedure of culturing human pluripotent stem cells (hPSCs) on inactivated mouse embryonic fibroblasts (MEFs).
Thawing of mouse embryonic fibroblasts (MEFs) for hPSC cultures
By savannah onThis protocol describes the thawing of mouse embryonic fibroblasts (MEFs) as feeder cells for human pluripotent stem cell (hPSC) culture.
Thawing of hPSCs grown on MEFs
By savannah onThis protocol describes the standard procedure of thawing human pluripotent stem cells (hPSCs) on inactivated mouse embryonic fibroblasts (MEFs).
Thawing of feeder-free hPSCs
By savannah onThis protocol describes the procedure of thawing feeder-free human pluripotent stem cells (hPSCs) using mTeSR-plus or StemFlex.
Subcloning of genotype-confirmed hPSCs clones
By savannah onThis protocol describes a standard procedure for subcloning of genotype-confirmed human pluripotent stem cells (hPSCs).
Standard operating procedure: Mouse transcardiac perfusion protocol
By savannah onMouse transcardiac perfusion protocol and storage.
Standard operating procedure: Mouse stereotaxic intracranial injection surgery
By savannah onThe intent of this standard operating procedure (SOP) is to describe procedures for mouse stereotaxic intracranial injection surgery.
Standard operating procedure for the isolation of genetically engineered hPSCs clones in a high-throughput way
By savannah onThis collection describes a standard procedure for isolating single human pluripotent stem cell (hPSC) clones in a high-throughput way. This collection 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
Single cell survival assay
By savannah onThis protocol describes the experimental procedure used to measure single cell survival rates post nucleofection of human pluripotent stem cells (hPSCs).
Seeding nucleofected hPSCs in 96-well plates using limited dilution
By savannah onThis protocol describes a standard procedure for seeding nucleofected human pluripotent stem cells (hPSCs) 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
Sample preparation for aCGH karyotyping
By savannah onThis protocol describes the standard procedure preparing cell pellets from human pluripotent stem cells (hPSCs) cultured on MEFs for outsourced aCGH karyotyping.