Computational protocol: Gain of BDNF Function in Engrafted Neural Stem Cells Promotes the Therapeutic Potential for Alzheimer’s Disease

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Protocol publication

[…] The experimental procedure and measurements are similar to that used in previous studies. Briefly, 4 weeks after control- or BDNF-NSCs transplantation, 150-μm-thick acute brain slices were prepared and incubated with 2 μM calcium orange-AM (Thermo) and 0.02% Pluronic F-127 (Thermo) for 1 h in aCSF and washed for 30 min at 37 °C with oxygen supplementation. During calcium imaging experiments, the temperature was maintained at 37 °C with sustained oxygen supplementation using a specialized chamber (GE Healthcare, Buckinghamshire, UK), and a DeltaVision Elite image system (GE Healthcare) equipped with an inverted microscope (Olympus IX71) and a 40× A-UPO oil-immersion objective (Olympus). Calcium orange indicator was excited at 542 nm and was emitted at 597 nm. The engrafted NSCs were identified by 475 nm excitation (GFP). In the collection of calcium fluctuations, during the first 200 seconds, the spontaneous [Ca2+]i oscillations were collected with no additional chemo transmitters or inducers, and the responses of calcium fluctuations were measured after treatment with 90 mM potassium chloride (Sigma). The collected images were stacked and transferred to grey scale or ratio-metric images using MetaMorph image analysis software (Moleculardevises), and the quantification of calcium fluctuations was measured with ImageJ software using the tool that sets the region of interest and multiple measurements. […]

Pipeline specifications

Software tools MetaMorph, ImageJ
Application Microscopic phenotype analysis
Diseases Alzheimer Disease, Neurodegenerative Diseases