Protocol detail

Differentiation of NPCs to Glutamatergic Neurons

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A total of 4 × 10⁴ NPCs were seeded on Matrigel-coated covers and differentiated to glutamatergic neurons. Cells were fixed in 4% paraformaldehyde and processed as previously described [18 (link)]. Immunofluorescence was performed using specific antibodies: rabbit anti microtubule-associated protein 2, Map2 (1:400; AB5622, Sigma-Aldrich); guinea pig anti vesicular glutamate transporter 1, Vglut1 (1:200; 135304, Synaptic System, Goettingen, Germany); Alexa fluor 488 donkey anti rabbit IgG (1:800; IS20015, Immunological Sciences, Roma, Italy); Alexa fluor 594 goat anti guinea pig IgG (1:800; A11076, ThermoFisher Scientific, Monza, Italy). Images were acquired by fluorescence microscope Zeiss Axio Observer.Z1 equipped with Hamamatsu EM-CCD 9100-02 camera and Volocity acquisition software.

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Santambrogio P., Ripamonti M., Paolizzi C., Panteghini C., Carecchio M., Chiapparini L., Raimondi M., Rubio A., Di Meo I., Cozzi A., Taverna S., De Palma G., Tiranti V, & Levi S. (2020). Harmful Iron-Calcium Relationship in Pantothenate kinase Associated Neurodegeneration. International Journal of Molecular Sciences, 21(10), 3664.

Publication 2020

AB5622 IS20015 A11076 Axio Observer.Z1 EM-CCD 9100-02 camera

Alexa 594 Alexa fluor 488 Anti igg Antibodies Cells Donkey Fluorescence microscope Goat Guinea pig Immunofluorescence Map2 Matrigel Microtubule associated protein 2 Neurons Paraformaldehyde Rabbit Roma Vesicular glutamate transporter 1

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Variable analysis

independent variables

NPC seeding density (4 × 10^4 NPCs)

dependent variables

Glutamatergic neuron differentiation

control variables

Matrigel coating of covers
Fixation in 4% paraformaldehyde
Immunofluorescence staining using specific antibodies (rabbit anti-Map2, guinea pig anti-Vglut1, Alexa Fluor 488 and 594 secondary antibodies)
Microscopy setup (Zeiss Axio Observer.Z1, Hamamatsu EM-CCD 9100-02 camera, Volocity acquisition software)

controls

Positive control: Not explicitly mentioned
Negative control: Not explicitly mentioned

Annotations

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