All animal preparations adhered to protocols approved by the Institutional Animal Care and Use Committee of the Veterans Administration Connecticut Healthcare System. DRG neurons were isolated from neonatal Sprague–Dawley rats and transfected with HaloTag-NaV1.8 as previously described (Dib-Hajj et al., 2009 (link); Higerd-Rusli et al., 2022 (link)). Studies of channel traffic used two microfluidic chambers separated by a physical barrier 450 μm in width with microgrooves to allow axons to extend from a soma chamber to the axon chamber (Xona Microfluidics, Research Triangle Park, NC) that were adhered to 50 mm glass-bottom dishes previously coated with poly-L-lysine and laminin as described (Akin et al., 2021 (link)). Transfected DRG neurons were applied to the somatic chambers and the growth medium (Neurobasal with 2% B27, 1% penicillin/streptomycin, 1% GlutaMAX, all from Thermo Fisher Scientific, Waltham, MA) was supplemented with 50 ng/ml NGF (Envigo, Indianapolis, IN) and GDNF (Preprotech, Windsor, NJ) the following day. The distal axonal chamber received medium with 100 ng/ml NGF and GDNF to attract growth of axons into this chamber. Cultures were treated as described below and analyzed on day 7 after preparation unless otherwise noted.
Nerve growth factor (ngf)
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NGF is a neurotrophin that promotes the survival and differentiation of neurons. It is a naturally occurring protein involved in the maintenance and growth of nerve cells.
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4 protocols using nerve growth factor (ngf)
1
DRG Neuron Culture with Microfluidics
2
In Vitro Wallerian Degeneration of Murine DRG Axons
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Murine DRG explants dissected from embryonic day 13 C56BL/6J mice were cultured on poly-l -lysine– and laminin-coated 24-well plates in Neuro medium (Miltenyi Biotec) containing 10% FBS and 25 ng/ml NGF (Envigo). After 24 h, the culture medium was changed to Neuro medium supplemented with 2% Neuro-Brew-21 (Miltenyi Biotec), 25 ng/ml NGF, and 1 mM glutamine in addition to a mixture of 1 mM 5′-fluoro-2′-deoxyuridine and 1 mM uridine to remove nonneuronal cells. The in vitro Wallerian degeneration of axons was introduced by removing cell bodies at 10–14 d in vitro using a pipette tip (Wakatsuki et al., 2011 (link), 2015 (link)).
3
Immortalization and Differentiation of Primary Brown Adipocytes
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Primary brown adipocytes were immortalized and differentiated in culture as previously described 47 (link). Briefly, confluent cells were incubated with induction medium (DMEM containing 10% FBS, 0.5 μΜ isobutylmethylxanthine (IBMX), 125 μΜ indomethacin, 1 μΜ dexamethasone, 1 nM triiodothyronine (T3), and 20 nM insulin). After 48 h, the medium was replaced with maintenance medium (DMEM supplemented with 10% FBS, 1 nM T3, and 20 nM insulin). For TSK treatments, recombinant human TSK protein (3940-TS; R&D Systems) was added to the induction medium at different concentrations. PC-12 cells were cultured in RPMI 1630 medium containing 10% horse serum and 5% FBS. For PC-12 cell differentiation, 40%-60% confluent cultures were exposed to RPMI 1640 containing 2% horse serum, 1% FBS and 100 ng/ml NGF (B5025, envigo). Fresh differentiation medium was added every two days until day 6.
4
Immortalization and Differentiation of Primary Brown Adipocytes
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