Computational protocol: Principles of branch dynamics governing shape characteristics of cerebellar Purkinje cell dendrites

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Protocol publication

[…] Purkinje cells from postnatal day (P) 0 mouse cerebella were dissociated and plated on a glass-based dish coated with poly-d-lysine in DMEM/F12 supplemented by 10% FBS. Two hours after plating, media were replaced by maintenance medium containing DMEM/F12, 0.1 mg/ml bovine serum albumin, 2.1 mg/ml glucose, 3.9 mM glutamine, a modified N3 supplement (8 μM progesterone, 20 μg/ml insulin, 100 μM putresine, 30 nM selenium dioxide) and 1% penicillin-streptomycin. Plasmids containing PKD1-K618N and TdTomato were transfected by nucleofection of dissociated cells from one and a half cerebella using 10 μg of each plasmid with nucleofector protocol O-03 (). Transfected cells were observed using a confocal microscope (FV1000, Olympus) with 40× air UPlan SApo objective. For time-lapse imaging, Purkinje cells were GFP-labeled by adding 1 μl of AAV-CAG-EGFP [109-1010 plaque-forming units (pfu)/ml] to the medium. Labeled cells were observed every 3 hours with an incubation microscope (LCV100, Olympus) using a 20× objective (numerical aperture 0.7, Olympus). Dendrites were traced and reconstructed with the aid of Neurolucida software (MBF Bioscience) for quantitative analysis using Neurolucida Explore and ImageJ (NIH). Branch angle was defined as the angle between the lines produced by a branch point and the branch points or dendritic tips terminating each of the daughter segments. All data are expressed as mean ± s.e.m. unless otherwise indicated. Comparisons of variables between two groups were made by Student’s t-test. A probability value of P<0.05 was considered to be significant. […]

Pipeline specifications

Software tools Neurolucida, ImageJ
Applications Laser scanning microscopy, Microscopic phenotype analysis
Diseases Keratitis, Dendritic