Postnatal-dissociated cortical neuron cultures were prepared as previously described (31 , 36 (link)), from newborn B6 mice. Neurons were plated into 35 mm glass bottom Petri dishes at a 1 million/ml density in culture medium consisting of Basal Medium Eagle supplemented with 10% bovine calf serum and 1% penicillin–streptomycin. On day in vitro 2 (DIV2), culture medium was changed to Neurobasal A medium, supplemented with B27-plus reagent (Invitrogen), Glutamax, and 1% penicillin–streptomycin. Neurons were transfected with the CryBAR optogenetic system (6 μg plasmid/plate) on DIV5 using Lipofectamine LTX reagent. On DIV7, culture medium was removed and neurons were placed in imaging solution (Mg-free HEPES-buffered artificial cerebrospinal fluid (37 (link))) Live-cell imaging was performed before and after illumination using a Leica DMi8 Live Cell Imaging System. Animal use protocols were approved by East Carolina University Institutional Animal Care and Use Committee.
B27 plus reagent
Manufactured by Thermo Fisher Scientific
The B27-plus reagent is a serum-free, protein-rich medium supplement designed to support the growth and survival of neuronal and other cell types in culture. It provides a combination of vitamins, hormones, and other essential components to promote optimal cell growth and development.
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2 protocols using b27 plus reagent
1
Optogenetic Stimulation of Cortical Neurons
2
Optogenetic Stimulation of Postnatal Cortical Neurons
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Postnatal dissociated cortical neuron cultures were prepared as previously described (Bunner, Dodson, Szatmari, & Hughes, 2021; Chang et al., 2017) (link), from newborn B6 mice. Neurons were plated into 35 mm glass bottom Petri dishes at a 1 million/ml density in culture medium consisting of BME supplemented with 10% BCS and 1% Penicillin-Streptomycin. On day in vitro 2 (DIV2), culture medium was changed to Neurobasal A medium, supplemented with B27-plus reagent (Invitrogen), Glutamax and 1% Penicillin-Streptomycin. Neurons were transfected with the CryBAR optogenetic system (6 µg plasmid/plate) on DIV5 using Lipofectamine LTX reagent (Invitrogen). On DIV7, culture medium was removed and neurons were placed in imaging solution (Mg-free HEPES buffered aCSF (Sun, Smirnov, Kamasawa, & Yasuda, 2021 (link))) Live cell imaging was performed before and after illumination using a Leica DMi8 Live Cell Imaging System.
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