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Live Cell Quantification using Image Analysis Resource Report Resource Website |
Minjung Song, Florian Merkle | DOI:10.17504/protocols.io.bpmcmk2w | Neurodegeneration Method Development Community | PurposeThis protocol describes image-based quantification of human pluripotent stem cells (hPSCs), which could be adapted for other cell populations that grow in a monolayer. It utilises a membrane-bound stain and requires a high-content imaging system such as a PerkinElmer Oprea Phenix or similar and downstream image analysis pipelines. The suggested dye labels live cells, fluoresces in far red, and shows no evidence of toxicity in hPSCs. This protocol is useful in situations where cell quantification by manual or automated counting becomes rate-limiting, such as when multiple lines are grown for high-throughput differentiation experiments on different genetic backgrounds, or for survival or morphology-based screens in systems where cells have clearly defined cell borders. | Wellcome-MRC Institute of Metabolic Science, University of Cambridge, Cambridge, CB2 0QQ, United Kingdom, Wellcome-MRC Institute of Metabolic Science, University of Cambridge, Cambridge, CB2 0QQ, United Kingdom | 1 | 2020 | Minjung Song, Florian Merkle 2020. Live Cell Quantification using Image Analysis. protocols.io https://dx.doi.org/10.17504/protocols.io.bpmcmk2w | 2021-04-15 09:15:03 | |||
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TRANSFECTION OF i3NEURONS (Support Protocol 3) Resource Report Resource Website |
Michael S. Fernandopulle, Ryan Prestil, Christopher Grunseich, Chao Wang, Li Gan, Michael E. Ward | DOI:10.17504/protocols.io.5wug7ew | Neurodegeneration Method Development Community | Transient protein expression can easily be studied in i3Neurons using lipid-based transfection. This protocol is identical to that in iPSCs (see Basic Protocol 2). i3Neurons are modestly transfectable, with 5 % to 10 % of cells showing fluorescent protein expression after 24 hr. We have found that refreshing neuronal medium 1 to 2 hr after transfection both allows successful DNA entry into cells and largely prevents cytotoxicity resulting from the transfection reagent. Unlike iPSCs, i3Neurons show increased protein expression/accumulation over time, with greater fluorescence 48 to 72 hr after transfection than at 24 hr. Transient transfections also show more durable expression in i3Neurons than iPSCs, likely because episomes are not diluted by cell division. i3Neurons can be transfected in suspension (i.e., re-plating after day 3 of differentiation) or as an adherent culture, although better results are observed in adherent cultures. They are also amenable to serial transfections (i.e., re-transfecting with the same construct 24 hr apart) if higher-percentage transfections are desired. | National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, Maryland, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, Maryland, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, Maryland, Gladstone Institute of Neurological Disease, Gladstone Institutes, San Francisco, California, Gladstone Institute of Neurological Disease, Gladstone Institutes, San Francisco, California, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, Maryland | https://doi.org/10.1002/cpcb.51 | 1 | 2019 | Michael S. Fernandopulle, Ryan Prestil, Christopher Grunseich, Chao Wang, Li Gan, Michael E. Ward 2019. TRANSFECTION OF i3NEURONS (Support Protocol 3) . protocols.io https://dx.doi.org/10.17504/protocols.io.5wug7ew | 2021-04-15 09:15:04 | ||
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RNA Imaging with MERFISH - Probe Construction Resource Report Resource Website |
Jeffrey R. Moffitt, Xiaowei Zhuang | DOI:10.17504/protocols.io.meqc3dw | Human Cell Atlas Method Development Community, Neurodegeneration Method Development Community | Staining hundreds to thousands of RNA species each of which requires tens of unique encoding probes requires a very large number of unique oligonucleotide sequences. The sheer number of unique sequences makes traditional solid phase oligonucleotide synthesis prohibitively expensive in most cases. For example, assuming a modest cost of $0.10/base, an encoding probe length of ~100 nt, and the need for ~50,000 unique oligos for a single MERFISH measurement, the cost of the needed oligos would be $500,000! To circumvent this astronomical cost, we have developed a high-throughput approach to generating these probes which utilizes array-derived synthesis of complex oligonucleotide pools. These arrayderived complex oligonucleotide pools that contain ~100,000 custom designed sequences can be purchased for only a few thousand dollars. The challenge to using these pools is that each individual sequence is provided in quantities far too small to be used directly for labeling. Thus, we developed an enzymatic amplification protocol to generate the encoding probes in high quantity sufficient for RNA FISH experiments using the array-derived complex oligonucleotide pools as templates. | Harvard University, Cambridge, Massachusetts, 02138 USA, Harvard University, Cambridge, Massachusetts, 02138 USA | https://www.ncbi.nlm.nih.gov/pubmed/27241748 | 1 | 2018 | Jeffrey R. Moffitt, Xiaowei Zhuang 2018. RNA Imaging with MERFISH - Probe Construction. protocols.io https://dx.doi.org/10.17504/protocols.io.meqc3dw | 2021-04-15 09:15:08 | ||
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Single Nuclei extraction from frozen brain Resource Report Resource Website |
Bruno Benitez | DOI:10.17504/protocols.io.2a5gag6 | Neurodegeneration Method Development Community, Functional Genomics | Protocol: Purification of nuclei from human brains using an iodixanol gradientUse BSL2 precautions. Samples are not known to be infective, but should be treated as such. Gloves, mask if outside hood, disposable gown. Every surface and reusable equipment (homogenizers, centrifuge tubes) washed with 25% bleach.Discarded supernatants or other tissue byproducts should go into 10% bleach for sterilization.Discarded tubes and tips go into biohazard waste stream.Solutions:Stock optiprep 60% Iodixanol [Sigma-Aldrich D1556-250mL]Dilutant:Final Concentrations: 900 mM KCL; 30 mM MgCl2; 120 mM Tricine-KOH (pH 7.8)Recipe for 50 mls1) 22.5 ml of 2M KCL, +2) 1.5 ml of 1M MgCl2, + 3) 6 ml of 1 M Tricine-KOH 4) brought up to 50 ml with molecular biology grade water. Components1) Tricine-KOH is 17.92 grams Tricine (Sigma T0377) and 100 mL RNAse free water for 1 M solution (pH to 7.8 with KOH as needed. Usually takes about 3-4 mL of 3M KOH).2) 1 M MgCl2 (AM9530G) and 2M KCl (AM9460G) are commercial RNAse free stocks from Ambion. Homogenization medium (need 6 ml per sample + ~ 2ml extra for 30% Iodixanol): Recipe (stable at 4C) for 100 mls / 50 mls1) .25 M Sucrose (8.56 g / 4.28g ) 2) 150 mM KCL (7.5 ml / 3.75of 2M)3) 5 mM MgCl2 (.5 ml / .25 ml of 1M)4) 20 mM Tricine-KOH (2 ml / 1 ml of 1M)5) ddH2O up to 100 ml /50 ml in RNAse free H20Supplementation (right before use): 1) For 4 samples make 35 mls of homogenization buffer and supplement with:1. 3.5 ul spermine, 2. 3.5 ul spermidine, 3. 35 ul RNASIN 4. EDTA free protease inhibitor tablet. Iodixanol Dilutions:50% Iodixanol 1) Recipe: 5 volumes stock (60%) optiprep + 1 volume of dilutant. 2) Scale: need 2 ml per sample tube + 3 ml per sample for 35% Iodixanol3) Example: 4 samples add 17.5 ml stock to 3.5 ml diluant.35% Iodixanol 1) Recipe: 7 ml 50% Iodixanol + 3 ml supplemented homogenization medium.2) Scale: need 4 ml per sample3) For 4 samples add 5.14 mL supplemented homogenization buffer to 12 mL of 50% Iodixanol.Resupsension Buffer for Nuclei Wash (new from 10x Genomics recipe)1) 22.5 ml of 2M KCL, +2) 1.5 ml of 1M MgCl2, + 3) 6 ml of 1 M Tricine-KOH 4) brought up to 50 ml with molecular biology grade water. Components1) Tricine-KOH is 17.92 grams Tricine (Sigma T0377) and 100 mL RNAse free water for 1 M solution (pH to 7.8 with KOH as needed. Usually takes about 3-4 mL of 3M KOH).2) 1 M MgCl2 (AM9530G) and 2M KCl (AM9460G) are commercial RNAse free stocks from Ambion. Homogenization medium (need 6 ml per sample + ~ 2ml extra for 30% Iodixanol): Recipe (stable at 4C) for 100 mls / 50 mls1) .25 M Sucrose (8.56 g / 4.28g ) 2) 150 mM KCL (7.5 ml / 3.75of 2M)3) 5 mM MgCl2 (.5 ml / .25 ml of 1M)4) 20 mM Tricine-KOH (2 ml / 1 ml of 1M)5) ddH2O up to 100 ml /50 ml in RNAse free H20Supplementation (right before use): 1) For 4 samples make 35 mls of homogenization buffer and supplement with:1. 3.5 ul spermine, 2. 3.5 ul spermidine, 3. 35 ul RNASIN 4. EDTA free protease inhibitor tablet. Iodixanol Dilutions:50% Iodixanol 1) Recipe: 5 volumes stock (60%) optiprep + 1 volume of dilutant. 2) Scale: need 2 ml per sample tube + 3 ml per sample for 35% Iodixanol3) Example: 4 samples add 17.5 ml stock to 3.5 ml diluant.35% Iodixanol 1) Recipe: 7 ml 50% Iodixanol + 3 ml supplemented homogenization medium.2) Scale: need 4 ml per sample3) For 4 samples add 5.14 mL supplemented homogenization buffer to 12 mL of 50% Iodixanol.Resupsension Buffer for Nuclei Wash (new from 10x Genomics recipe)1) Tricine-KOH is 17.92 grams Tricine (Sigma T0377) and 100 mL RNAse free water for 1 M solution (pH to 7.8 with KOH as needed. Usually takes about 3-4 mL of 3M KOH).2) 1 M MgCl2 (AM9530G) and 2M KCl (AM9460G) are commercial RNAse free stocks from Ambion.Homogenization medium (need 6 ml per sample + ~ 2ml extra for 30% Iodixanol): Recipe (stable at 4C) for 100 mls / 50 mls1) .25 M Sucrose (8.56 g / 4.28g ) 2) 150 mM KCL (7.5 ml / 3.75of 2M)3) 5 mM MgCl2 (.5 ml / .25 ml of 1M)4) 20 mM Tricine-KOH (2 ml / 1 ml of 1M)5) ddH2O up to 100 ml /50 ml in RNAse free H20Supplementation (right before use): 1) For 4 samples make 35 mls of homogenization buffer and supplement with:1. 3.5 ul spermine, 2. 3.5 ul spermidine, 3. 35 ul RNASIN 4. EDTA free protease inhibitor tablet. Iodixanol Dilutions:50% Iodixanol 1) Recipe: 5 volumes stock (60%) optiprep + 1 volume of dilutant. 2) Scale: need 2 ml per sample tube + 3 ml per sample for 35% Iodixanol3) Example: 4 samples add 17.5 ml stock to 3.5 ml diluant.35% Iodixanol 1) Recipe: 7 ml 50% Iodixanol + 3 ml supplemented homogenization medium.2) Scale: need 4 ml per sample3) For 4 samples add 5.14 mL supplemented homogenization buffer to 12 mL of 50% Iodixanol.Resupsension Buffer for Nuclei Wash (new from 10x Genomics recipe)1) .25 M Sucrose (8.56 g / 4.28g ) 2) 150 mM KCL (7.5 ml / 3.75of 2M)3) 5 mM MgCl2 (.5 ml / .25 ml of 1M)4) 20 mM Tricine-KOH (2 ml / 1 ml of 1M)5) ddH2O up to 100 ml /50 ml in RNAse free H20Supplementation (right before use): 1) For 4 samples make 35 mls of homogenization buffer and supplement with:1. 3.5 ul spermine, 2. 3.5 ul spermidine, 3. 35 ul RNASIN 4. EDTA free protease inhibitor tablet. Iodixanol Dilutions:50% Iodixanol 1) Recipe: 5 volumes stock (60%) optiprep + 1 volume of dilutant. 2) Scale: need 2 ml per sample tube + 3 ml per sample for 35% Iodixanol3) Example: 4 samples add 17.5 ml stock to 3.5 ml diluant.35% Iodixanol 1) Recipe: 7 ml 50% Iodixanol + 3 ml supplemented homogenization medium.2) Scale: need 4 ml per sample3) For 4 samples add 5.14 mL supplemented homogenization buffer to 12 mL of 50% Iodixanol.Resupsension Buffer for Nuclei Wash (new from 10x Genomics recipe)1) For 4 samples make 35 mls of homogenization buffer and supplement with:1. 3.5 ul spermine, 2. 3.5 ul spermidine, 3. 35 ul RNASIN 4. EDTA free protease inhibitor tablet. Iodixanol Dilutions:50% Iodixanol 1) Recipe: 5 volumes stock (60%) optiprep + 1 volume of dilutant. 2) Scale: need 2 ml per sample tube + 3 ml per sample for 35% Iodixanol3) Example: 4 samples add 17.5 ml stock to 3.5 ml diluant.35% Iodixanol 1) Recipe: 7 ml 50% Iodixanol + 3 ml supplemented homogenization medium.2) Scale: need 4 ml per sample3) For 4 samples add 5.14 mL supplemented homogenization buffer to 12 mL of 50% Iodixanol.Resupsension Buffer for Nuclei Wash (new from 10x Genomics recipe)Iodixanol Dilutions:50% Iodixanol 1) Recipe: 5 volumes stock (60%) optiprep + 1 volume of dilutant. 2) Scale: need 2 ml per sample tube + 3 ml per sample for 35% Iodixanol3) Example: 4 samples add 17.5 ml stock to 3.5 ml diluant.35% Iodixanol 1) Recipe: 7 ml 50% Iodixanol + 3 ml supplemented homogenization medium.2) Scale: need 4 ml per sample3) For 4 samples add 5.14 mL supplemented homogenization buffer to 12 mL of 50% Iodixanol.Resupsension Buffer for Nuclei Wash (new from 10x Genomics recipe)1) Recipe: 5 volumes stock (60%) optiprep + 1 volume of dilutant. 2) Scale: need 2 ml per sample tube + 3 ml per sample for 35% Iodixanol3) Example: 4 samples add 17.5 ml stock to 3.5 ml diluant.35% Iodixanol 1) Recipe: 7 ml 50% Iodixanol + 3 ml supplemented homogenization medium.2) Scale: need 4 ml per sample3) For 4 samples add 5.14 mL supplemented homogenization buffer to 12 mL of 50% Iodixanol.Resupsension Buffer for Nuclei Wash (new from 10x Genomics recipe)1) Recipe: 7 ml 50% Iodixanol + 3 ml supplemented homogenization medium.2) Scale: need 4 ml per sample3) For 4 samples add 5.14 mL supplemented homogenization buffer to 12 mL of 50% Iodixanol. i. 1x PBS with 1.0% BSA and 0.2 U/uL1) Recipe:___________ commercial PBS (46-013CM 10x PBS from Corning)2) Recipe: __________ BSA (001000162 from Jackson Immunoresearch)3) Recipe: ____1:1000______ RNAsinProtocol: 1. Turn on the Beckman Allegra 64R, and Eppendorf 5430R Centrifuges so they can get to 4o. i. For Beckman put in swing-out rotor (S0410).***Make sure acceleration and deceleration are set to 0 on Beckman*** 2. Supplement the homogenization medium. i. Add fresh .15mM spermine (1:10,000) (1 ul per 10 ml) ii. Add fresh .5mM spermidine (1:10,000) (1 ul per 10 ml) iii. Add 1 µl RNASIN per ml of homogenization medium if using for RNA iv. Add EDTA free protease inhibitor tablet 3. Prepare working concentrations of rest of solutions.4. Aliquot 2ml of supplemented homogenization medium per 15 ml homogenization tube for each brain. We have used 1g of tissue successfully to yield 15-20 million nuclei. We also used 2g and double that. 5. Check weight of brain sample and keep on ice in 2ml supplemented homogenization medium.6. Homogenize the brain tissue in the 2 ml supplemented homogenization medium. i. Be careful at all steps to treat all samples exactly equally with regard to handling, as different cell type’s nuclei might be more or less vulnerable to physical destruction. All pipetting and resuspension steps should be gentle and consistent to avoid shearing and clumping of nuclei. ii. Put tissue into 15 ml Kontes glass homogenizer tubes iii. Homogenize 10 strokes on ice with ‘A’, and 10 strokes on ice with ‘B’ pestle. i. Use in BSL2 space for human tissue (currently using chem fume hood, with diapers). iv. Check to see that no large pieces have escaped homogenization. v. Transfer pipette with 2ml strippette the homogenate equally into 2 x 2 ml siliconized snap cap microcentrifuge tubes. vi. Add another 500ul supplemented homogenization buffer to the glass homogenizer and transfer remaining homogenate to the same microcentrofuge tubes. i. After use with human tissue, decontaminate homogenizers with 10% bleach during a break in the protocol. Dispose of diapers, tips, etc, in BSL2 waste. ii. Then RNASe Zap, then rinse with ethanol then ddh20 water. 7. Spin tubes at low speed (500 RCF) in the Eppendorf for 5 minutes at 4o.8. Pour off the supernatant, add 1 ml supplemented homogenization medium, and turn over or flick to resuspend. i. Take 100 ul of supernatant and freeze at -80 if you want cytoplasmic RNA later9. Repeat step 8.10. While spinning you can add 4ml of 35% Iodixanol to a centrifuge tube for each sample. *Mix the solution up and down to mix again after it has been sitting* i. These are polycarbonate centrifuge tubes from Beckman Coulter, stored in cabinet under the Sorvall in the shared equipment room. ii. May need to do a quick spin of the 35% to get it to settle down if some is stuck on the walls.11. Resuspend each pellet in 700 uls of supplemented homogenization buffer. Combine all tubes for each sample into a single 15 ml conical. 12. Add an equal volume of 50% Iodixanol to the samples in the 15 ml conicals. This will create a final 25% Iodixanol. *Mix 50% Iodixanol right before usage*13. Pipette the samples up and down with low retention pipette tips and carefully layer the ~ 3ml-4mls of 25% Iodixanol (2 tubes) on top of the 4 mL of 35% Iodixanol using a low attachment tip (silver box). You will need to use the same volume of all samples so that tubes are balanced, so scale to your smallest sample. i. Layering is best done by holding the bottle almost horizontal and using a P1000 to pipette slowly into the wall of the tube slightly above the 35 % Iodixanol. It is important to make sure the layers do not mix. 14. Centrifuge the tubes at 10,000 rpm (top speed) for 30 minutes in the Beckman Allegra *Check acceleration and deceleration again*15. Carefully remove the centrifuge tubes. Take back to BSL2 space. The nuclei should be visible as a cloudy layer in the interface of the 25%/35% Iodixanol. with a transfer pippette, remove the top pink debris layer of myelin (suck off with a transfer pipette).16. With a low retention pipette remove the nuclei by vacuuming them up from just above the layer. Then place into a 1.5mL Eppendorf – NONSTICK TUBES (for nuclei). No more than 500 uls per tube. Usually 2 tubes per sample. 17. Add 1 ml of supplemented Nuclei Wash buffer to the tubes & mix. 18. Centrifuge at 500g’s (rcf) at 4o.19. Nuclei should be pelleted at the bottom20. Remove & discard supernatant 21. Resuspend nuclei in about 750 mls of Nuclei Wash buffer. Bring together all nuclei from the same sample into a single tube.22. Centrifuge at 500g’s (rcf) at 4o.23. Nuclei should be pelleted at the bottom24. Remove & discard supernatant 25. Resuspend nuclei in about 1000 mls of Nuclei Wash buffer26. Remove & discard supernatant 27. Resuspend nuclei in about 1000 ul of Nuclei Wash buffer. 28. Let it drip through a 40 um Nylon mesh cell strainer (#229481)to remove clumps per 10x protocol.29. Take out 20 uls, mix 1:1 with a 1:2000 DAPI solution, and count on hemacytometer or countess and confirm lack of clumps.30. Adjust aliquot of nuclei to desired dilution (1,200 cells per ul). Count again to confirm dilution.31. The rest may be stored as follows: Pellet and resuspend in 100% methanol | Washington University, Saint Louis | 1 | 2019 | Bruno Benitez 2019. Single Nuclei extraction from frozen brain. protocols.io https://dx.doi.org/10.17504/protocols.io.2a5gag6 | 2021-04-15 09:15:34 | |||
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Footprint-Free Genome Editing of iPSC Using Alt-R CRISPR/Cas9 Resource Report Resource Website |
Jacob Marsh, Rj Martinez, Celeste Karch | DOI:10.17504/protocols.io.bfmmjk46 | Neurodegeneration Method Development Community | Washington University in Saint Louis - WUSTL (MO), Washington University in St Louis, Washington University in St Louis | 1 | 2020 | Jacob Marsh, Rj Martinez, Celeste Karch 2020. Footprint-Free Genome Editing of iPSC Using Alt-R CRISPR/Cas9. protocols.io https://dx.doi.org/10.17504/protocols.io.bfmmjk46 | 2021-04-15 09:15:11 | ||||
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ISOLATION AND VALIDATION OF CLONAL TRANSGENIC LINES (Basic Protocol 4) Resource Report Resource Website |
Michael S. Fernandopulle, Ryan Prestil, Christopher Grunseich, Chao Wang, Li Gan, Michael E. Ward | DOI:10.17504/protocols.io.5f7g3rn | Neurodegeneration Method Development Community | While transgenic populations may be enriched to near-purity using the techniques described above, the derivation of a clonal iPSC line descended from a single parent cell is necessary for proper genotyping and for many downstream applications, and it is thus standard practice following any genetic edit. One common method for isolating such clones, as described below, is by serial dilutions on two 6-well plates, followed by manual picking. As with all single-cell dissociations of iPSCs, the use of Y-27632 ROCK inhibitor is required until colonies are properly established; wells with fewer cells may require treatment for 2 to 3 days. 6-well plates are used because their wells have a larger surface area than other multiwell dishes, facilitating downstream picking. For highly enriched populations, serial dilutions may be reduced to one 6-well plate loaded with 1 × 105 cells in the first well, as there is a higher probability of identifying a purely positive colony.Following isolation, this protocol further describes basic genotyping in parallel with the gradual expansion of clonal lines, utilizing QuickExtract to prepare genomic DNA and PCR to test for the presence of a transgene of interest. Primer sequences and other specifics are included in more detail for the particular differentiation cassettes in Support Protocol 1. In general, basic genotyping by PCR should be performed as quickly as possible to screen out negative clones, while potentially positive clones should be confirmed by more stringent methods such as Sanger sequencing and western blotting as appropriate. To save culture reagents, negative clones may be immediately discarded, while all others should be expanded and frozen pending confirmation. | National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, Maryland, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, Maryland, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, Maryland, Gladstone Institute of Neurological Disease, Gladstone Institutes, San Francisco, California, Gladstone Institute of Neurological Disease, Gladstone Institutes, San Francisco, California, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, Maryland | https://doi.org/10.1002/cpcb.51 | 1 | 2019 | Michael S. Fernandopulle, Ryan Prestil, Christopher Grunseich, Chao Wang, Li Gan, Michael E. Ward 2019. ISOLATION AND VALIDATION OF CLONAL TRANSGENIC LINES (Basic Protocol 4). protocols.io https://dx.doi.org/10.17504/protocols.io.5f7g3rn | 2021-04-15 09:15:39 | ||
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Picking Clones Resource Report Resource Website |
Celeste Karch, Rita Martinez, Jacob Marsh | DOI:10.17504/protocols.io.x77frrn | Neurodegeneration Method Development Community | Washington University in St Louis, Washington University in St Louis, Washington University in St Louis | 1 | 2019 | Celeste Karch, Rita Martinez, Jacob Marsh 2019. Picking Clones. protocols.io https://dx.doi.org/10.17504/protocols.io.x77frrn | 2021-04-15 09:15:39 | ||||
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Differentiation of NPC into cortical neurons Resource Report Resource Website |
Celeste Karch, Rita Martinez, Jacob Marsh | DOI:10.17504/protocols.io.x9bfr2n | Neurodegeneration Method Development Community | Washington University in St Louis, Washington University in St Louis, Washington University in St Louis | 1 | 2019 | Celeste Karch, Rita Martinez, Jacob Marsh 2019. Differentiation of NPC into cortical neurons. protocols.io https://dx.doi.org/10.17504/protocols.io.x9bfr2n | 2021-04-15 09:15:16 | ||||
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ASTROCYTE PRODUCTION (Support Protocol 7.1) Resource Report Resource Website |
Michael S. Fernandopulle, Ryan Prestil, Christopher Grunseich, Chao Wang, Li Gan, Michael E. Ward | DOI:10.17504/protocols.io.5xag7ie | Neurodegeneration Method Development Community | National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, Maryland, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, Maryland, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, Maryland, Gladstone Institute of Neurological Disease, Gladstone Institutes, San Francisco, California, Gladstone Institute of Neurological Disease, Gladstone Institutes, San Francisco, California, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, Maryland | https://doi.org/10.1002/cpcb.51 | 1 | 2019 | Michael S. Fernandopulle, Ryan Prestil, Christopher Grunseich, Chao Wang, Li Gan, Michael E. Ward 2019. ASTROCYTE PRODUCTION (Support Protocol 7.1). protocols.io https://dx.doi.org/10.17504/protocols.io.5xag7ie | 2021-04-15 09:15:43 | |||
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iPSC Differentiation Resource Report Resource Website |
Yingchao Xue, Xiping Zhan, Shisheng Sun, Senthilkumar S. Karuppagounder, Shuli Xia, Valina L Dawson, Ted M Dawson, John Laterra, Jianmin Zhang, Mingyao Ying | Neurodegeneration Method Development Community | Department of Immunology, Research Center on Pediatric Development and Diseases, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and School of Basic Medicine, Peking Union Medical College, State Key Laboratory of Medical Molecular Biology; Hugo W. Moser Research Institute at Kennedy Krieger, Department of Physiology and Biophysics, Howard University, College of Life Sciences, Northwest University, Department of Neurology, Johns Hopkins University School of Medicine; Neuroregeneration and Stem Cell Programs, Institute for Cell Engineering, Johns Hopkins University School of Medicine; Adrienne Helis Malvin Medical Research Foundation, Hugo W. Moser Research Institute at Kennedy Krieger; Department of Neurology, Johns Hopkins University School of Medicine, Department of Neurology, Johns Hopkins University School of Medicine; Neuroregeneration and Stem Cell Programs, Institute for Cell Engineering, Johns Hopkins University School of Medicine; Adrienne Helis Malvin Medical Research Foundation; Department of Neuroscience, Johns Hopkins University School of Medicine; Department of Physiology, Johns Hopkins University School of Medicine, Department of Neurology, Johns Hopkins University School of Medicine; Neuroregeneration and Stem Cell Programs, Institute for Cell Engineering, Johns Hopkins University School of Medicine; Adrienne Helis Malvin Medical Research Foundation; Department of Neuroscience, Johns Hopkins University School of Medicine; Department of Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine, Hugo W. Moser Research Institute at Kennedy Krieger; Department of Neurology, Johns Hopkins University School of Medicine; Department of Neuroscience, Johns Hopkins University School of Medicine; Department of Oncology, Johns Hopkins University School of Medicine, Department of Immunology, Research Center on Pediatric Development and Diseases, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and School of Basic Medicine, Peking Union Medical College, State Key Laboratory of Medical Molecular Biology, Hugo W. Moser Research Institute at Kennedy Krieger; Department of Neurology, Johns Hopkins University School of Medicine | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6344911/ | 1 | 2020 | Yingchao Xue, Xiping Zhan, Shisheng Sun, Senthilkumar S. Karuppagounder, Shuli Xia, Valina L Dawson, Ted M Dawson, John Laterra, Jianmin Zhang, Mingyao Ying 2020. iPSC Differentiation. protocols.io https:// | 2021-04-15 09:15:16 | ||||
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Splitting 96 Well Plates for gDNA Extraction and Freezing Down Resource Report Resource Website |
Celeste Karch, Rita Martinez, Jacob Marsh | DOI:10.17504/protocols.io.x79frr6 | Neurodegeneration Method Development Community | Washington University in St Louis, Washington University in St Louis, Washington University in St Louis | 1 | 2019 | Celeste Karch, Rita Martinez, Jacob Marsh 2019. Splitting 96 Well Plates for gDNA Extraction and Freezing Down. protocols.io https://dx.doi.org/10.17504/protocols.io.x79frr6 | 2021-04-15 09:15:19 | ||||
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Feeding 6wp Resource Report Resource Website |
Andrea Argouarch | DOI:10.17504/protocols.io.8gihtue | Neurodegeneration Method Development Community | Protocol for feeding dural cells after outgrowth from tissue in a 6 well plate. | University of California, San Francisco | 1 | 2020 | Andrea Argouarch 2020. Feeding 6wp. protocols.io https://dx.doi.org/10.17504/protocols.io.8gihtue | 2021-04-15 09:15:28 | |||
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RNA Imaging with MERFISH - Data Analysis Resource Report Resource Website |
Jeffrey R. Moffitt, Xiaowei Zhuang | DOI:10.17504/protocols.io.metc3en | Human Cell Atlas Method Development Community, Neurodegeneration Method Development Community | The basic decoding of MERFISH data—the identification of specific RNA species for each spot in the sample—consists of three basic steps. First, fluorescent spots must be identified in all images. Second, slight changes in the stage position between images of the same region from different hybridization rounds must be corrected. Finally, sets of fluorescent spots that occupy the same location in the sample must be decoded into binary barcodes, which, in turn, are decoded into the specific RNA species. After these RNAs have been decoded, there are a series of simple calculations that can often be performed on the measured barcodes to determine important parameters associated with the performance of the measurements, such as estimates of the binary errors made at each bit or the relative confidence that can be ascribed to the counts for each RNA species. In this section, we provide protocols for performing these basic computational tasks. Example software and data can be found at: http://zhuang.harvard.edu/merfish/. | Harvard University, Cambridge, Massachusetts, 02138 USA, Harvard University, Cambridge, Massachusetts, 02138 USA | https://www.ncbi.nlm.nih.gov/pubmed/27241748 | 1 | 2018 | Jeffrey R. Moffitt, Xiaowei Zhuang 2018. RNA Imaging with MERFISH - Data Analysis. protocols.io https://dx.doi.org/10.17504/protocols.io.metc3en | 2021-04-15 09:15:25 | ||
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Splitting 96 Well Plates for gDNA Extraction and Continuing Culture Resource Report Resource Website |
Celeste Karch, Rita Martinez, Jacob Marsh | DOI:10.17504/protocols.io.x78frrw | Neurodegeneration Method Development Community | Washington University in St Louis, Washington University in St Louis, Washington University in St Louis | 1 | 2019 | Celeste Karch, Rita Martinez, Jacob Marsh 2019. Splitting 96 Well Plates for gDNA Extraction and Continuing Culture. protocols.io https://dx.doi.org/10.17504/protocols.io.x78frrw | 2021-04-15 09:15:29 | ||||
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ExFISH Protocols For Cells and Tissues Resource Report Resource Website |
Fei Chen, Asmamaw T Wassie, Allison J Cote, Anubhav Sinha, Shahar Alon, Shoh Asano, Evan R Daugharthy, Jae-Byum Chang, Adam Marblestone, George M Church, Arjun Raj, Edward S Boyden | DOI:10.17504/protocols.io.n7bdhin | Human Cell Atlas Method Development Community, Neurodegeneration Method Development Community | These are Expansion FISH (ExFISH) protocols for super-resolution imaging of RNA structure and location with diffraction-limited microscopes.Please select the appropriate protocol depending on your sample (cells or tissue). | Massachusetts Institute of Technology, Massachusetts Institute of Technology, University of Pennsylvania, Massachusetts Institute of Technology, Massachusetts Institute of Technology, Massachusetts Institute of Technology, Harvard University, Massachusetts Institute of Technology, Massachusetts Institute of Technology, Harvard University, University of Pennsylvania, Massachusetts Institute of Technology | http://expansionmicroscopy.org | 1 | 2018 | Fei Chen, Asmamaw T Wassie, Allison J Cote, Anubhav Sinha, Shahar Alon, Shoh Asano, Evan R Daugharthy, Jae-Byum Chang, Adam Marblestone, George M Church, Arjun Raj, Edward S Boyden 2018. ExFISH Protocols For Cells and Tissues. protocols.io https://dx.doi.org/10.17504/protocols.io.n7bdhin | 2021-04-15 09:15:45 | ||
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LIPID-MEDIATED TRANSFECTION OF iPSCs (Basic Protocol 2) Resource Report Resource Website |
Michael S. Fernandopulle, Ryan Prestil, Christopher Grunseich, Chao Wang, Li Gan, Michael E. Ward | DOI:10.17504/protocols.io.5deg23e | Neurodegeneration Method Development Community | 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). | National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, Maryland, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, Maryland, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, Maryland, Gladstone Institute of Neurological Disease, Gladstone Institutes, San Francisco, California, Gladstone Institute of Neurological Disease, Gladstone Institutes, San Francisco, California, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, Maryland | https://doi.org/10.1002/cpcb.51 | 1 | 2019 | 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 https://dx.doi.org/10.17504/protocols.io.5deg23e | 2021-04-15 09:16:10 | ||
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Splitting p0 (6wp) to p1 (T75) Resource Report Resource Website |
Andrea Argouarch | DOI:10.17504/protocols.io.8gjhtun | Neurodegeneration Method Development Community | Protocol includes splitting outgrowth of dural cell from a 6 well plate into one T75 flask for expansion. | University of California, San Francisco | 1 | 2020 | Andrea Argouarch 2020. Splitting p0 (6wp) to p1 (T75). protocols.io https://dx.doi.org/10.17504/protocols.io.8gjhtun | 2021-04-15 09:16:17 | |||
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Characterization of iPSC Resource Report Resource Website |
Celeste Karch, Rita Martinez, Jacob Marsh | DOI:10.17504/protocols.io.x82frye | Neurodegeneration Method Development Community | Washington University in St Louis, Washington University in St Louis, Washington University in St Louis | 1 | 2019 | Celeste Karch, Rita Martinez, Jacob Marsh 2019. Characterization of iPSC . protocols.io https://dx.doi.org/10.17504/protocols.io.x82frye | 2021-04-15 09:16:27 | ||||
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DIFFERENTIATION OF i3NEURONS (Basic Protocol 5) Resource Report Resource Website |
Michael S. Fernandopulle, Ryan Prestil, Christopher Grunseich, Chao Wang, Li Gan, Michael E. Ward | DOI:10.17504/protocols.io.5h6g39e | Neurodegeneration Method Development Community | This protocol describes the rapid and robust differentiation of cortical neurons from hiPSCs via induced expression of the neurogenin-2 (NGN2) transcription factor (Wang et al., 2017; Zhang et al., 2013). To begin, iPSCs with a stably integrated human or mouse neurogenin-2 transgene under a tetracycline-inducible promoter are exposed to doxycycline in neuronal induction medium (IM). Since iPSCs grow as colonies, they must be single-cell dissociated to a new plate before doxycycline treatment in order to provide the differentiating neurons adequate space to begin producing neuritic extensions.Once the cells have been partially differentiated on Matrigel, they are re-plated onto dishes coated with poly-L-ornithine (PLO) for neuronal maturation. After 3 days of doxycycline treatment, the cells are committed to neural differentiation, although at this time they may have only minor neuritic elongations. These neurites are generally well-preserved after dissociation and replating, but the longer neuritic projections present in cells differentiated past 3 days are often damaged during the splitting process. For this reason, differentiated neurons are optimally replated on day 3. At this time, differentiating neurons can also be cryopreserved for use at a later date, enabling curation of large, standardized stocks of partially differentiated neurons for future experiments. | National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, Maryland, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, Maryland, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, Maryland, Gladstone Institute of Neurological Disease, Gladstone Institutes, San Francisco, California, Gladstone Institute of Neurological Disease, Gladstone Institutes, San Francisco, California, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, Maryland | https://doi.org/10.1002/cpcb.51 | 1 | 2019 | Michael S. Fernandopulle, Ryan Prestil, Christopher Grunseich, Chao Wang, Li Gan, Michael E. Ward 2019. DIFFERENTIATION OF i3NEURONS (Basic Protocol 5). protocols.io https://dx.doi.org/10.17504/protocols.io.5h6g39e | 2021-04-15 09:16:20 | ||
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Rapid Nuclei Isolation from Human Brain Resource Report Resource Website |
Fatma Ayhan, Genevieve Konopka | DOI:10.17504/protocols.io.scpeavn | Human Cell Atlas Method Development Community, Neurodegeneration Method Development Community | This protocol outlines our preparation of single-cell suspension from surgically acquired fresh human adult brain tissue. This method is adapted from Habib et al1.1 Habib, N. et al. Massively parallel single-nucleus RNA-seq with DroNc-seq. Nat Methods 14, 955-958, doi:10.1038/nmeth.4407 (2017). | Neurocrine Biosciences, UT Southwestern Medical Center | 1 | 2018 | Fatma Ayhan, Genevieve Konopka 2018. Rapid Nuclei Isolation from Human Brain. protocols.io https://dx.doi.org/10.17504/protocols.io.scpeavn | 2021-04-15 09:16:35 |
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