Cell type characterization of Boost and Boost+ mDA neuron protocol in vitro and in grafted in unilateral 6-hydroxydopamine (OHDA)-induced Parkinsonian rats
Cell type characterization of Boost and Boost+ mDA neuron protocol in vitro and in grafted in unilateral 6-hydroxydopamine (OHDA)-induced Parkinsonian rats
The tissue region of interest, the striatum, was grossly dissected out using a pre-chilled scalpel on ice and roughly minced into small chunks, and transferred using low-attachment 1,000p pipet by resuspending them in 1 mL of ice-cold homogenization buffer (HM). Tissue resuspension in a glass dounce homogenizer was gently homogenized with 10 strokes of the loose (A) pestle, followed by 20 strokes of the tight (B) pestle. The homogenized suspension was transferred to a pre-chilled low DNA-bind Eppendorf tube and centrifuged for 1,000g for 8 minutes at 4°C countertop microcentrifugation. The pellets were then gently resuspended in 250 µL HM buffer, which was then later mixed with 250 µL of 50% iodixanol mixture. During optiprep gradient system where 500 µL nuclei mixture was overlayed on top of 500 µL of 29% iodixanol solvent in a pre-chilled Eppendorf tube for high-speed centrifugation at 13,500g for 20 minutes at 4°C countertop microcentrifugation. The pellet is resuspended in nuclei storage buffer (NSB), counter-stained for DAPI, and processed for sorting to enrich human nuclei. The composition of each buffer was the following: HM is made from NIM2, 0.1% Triton-X 100, 40U/µL Rnasin, 20U/µL Superasin, and 5 µg /ml actinomycin D. Nuclei isolation medium 2 (NIM2) was made fresh for immediate use and discarded which is made up of NIM1, 0.5 mM DTT (dithiothreitol), and protease inhibitors (Roche mini complete, EDTA-free) 1 tablet for 10 mL solution. Nuclei isolation medium 1 (NIM1) was composed of 320 mM sucrose, 0.1 mM EDTA, 10 mM Tris buffer (pH 8.0), 5 mM MgCl2, 150 mM KCl, nuclease-free water (up to 100 mL). Iodixanol medium diluent (IDM) was made up of 250 mM sucrose, 150 mM KCl, 30 mM MgCl2, 10 mM Tris buffer (pH 8.0), and nuclease-free water (up to 100 mL). To prepare 29% and 50% iodixanol gradient, optiprep 60% iodixanol (D1556-250ml from Sigma) was diluted with IDM as solvent. Nuclei storage buffer (NSB) was 166.5 mM sucrose, 4 mM MgCl2, 10 mM Tris buffer (pH 8.0), 1 \% BSA, nuclease-free water (up to 100 mL), 5ug/ml actinomycin D, 40U/µL Rnasin, and 20U/µL Superasin. 10X chromium Single Cell 3' v3  10X chromium Single Cell 3' v3