Joy Guedia, Paola Brun, Sukhada Bhave, Sylvia Fitting, Minho Kang, William L. Dewey, Kurt F. Hauser & Hamid I. Akbarali. HIV-1 Tat exacerbates lipopolysaccharide-induced cytokine release via TLR4 signaling in the enteric nervous system. Scientific Reports 6, Article number: 31203 (2016) doi:10.1038/srep31203

...Myenteric neurons and glia were isolated as described recently41. Briefly, after euthanizing mice, the ileum was immediately removed and placed in ice-cold Krebs solution (118 mM NaCl, 4.6 mM KCl, 1.3 mM NaH2PO4, 1.2 mM MgSO4, 25 mM NaHCO3, 11 mM glucose and 2.5 mM CaCl2) bubbled with carbogen (95% O2/5% CO2). The ileum contents were discarded by passing Krebs solution using a syringe. The ileum was then divided into short segments which were threaded longitudinally on a plastic rod through the lumen and the longitudinal muscle containing the myenteric plexus (LMMP) strips were obtained using a cotton-tipped applicator. LMMP strips were rinsed three times in 1 ml Krebs and gathered by centrifugation (350 × g, 30 sec). LMMP strips were then minced with scissors and digested in 1.3 mg/ml collagenase type II (Worthington) and 0.3 mg/ml bovine serum albumin in bubbled Krebs (37 °C) for 1 h, followed by 0.05% trypsin for 7 min. Following each digestion, cells were triturated and collected by centrifuge (350 x g for 8 min). Cells were then plated on laminin (BD Biosciences) and poly-D-lysine coated coverslips in Neurobasal A media containing B-27 supplement, 1% fetal bovine serum, 10 ng/ml glial cell line-derived neurotrophic factor (GDNF, Neuromics, Edina, MN), and penicillin/streptomycin. Half of the cell media was replaced every 2–3 days with fresh complete neuron media...

Aravind R. Gade, Minho Kang, Fayez Khan, John.R. Grider, M.Imad Damaj, William L. Dewey and Hamid I. Akbarali. Enhanced sensitivity of α3β4 nicotinic receptors in enteric neurons after chronic morphine: implication for opioid-induced constipation.The Journal of Pharmacology and Experimental Therapeutics Published online before print April 11, 2016, doi: 10.1124/jpet.116.233304

... GDNF (glial cell-derived neurotrophic factor) was purchased from Neuromics (Edina, MN)...

S. Fitting, J. Ngwainmbi, M. Kang, F. A. Khan, D. L. Stevens, W. L. Dewey, P. E. Knapp, K. F. Hauser and H. I. Akbarali. Sensitization of enteric neurons to morphine by HIV-1 Tat protein. Neurogastroenterology & Motility. DOI: 10.1111/nmo.12514.

... Cells were then plated on laminin (BD Biosciences, San Jose, CA, USA) and poly-d-lysine coated coverslips in Neurobasal A media containing B-27 supplement, 1% fetal bovine serum, 10 ng/mL glial cell line-derived neurotrophic factor (Neuromics, Edina, MN, USA)...

Tricia H. Smith, John R. Grider, William L. Dewey, Hamid I. Akbarali. Morphine Decreases Enteric Neuron Excitability via Inhibition of Sodium Channels. PLoS ONE 7(9): e45251. doi:10.1371/journal.pone.0045251.

...All chemicals and reagents were obtained from Sigma Aldrich (St Louis, MO), unless otherwise noted, except cell culture reagents, which were purchased from Gibco (Grand Island, NY). Male Swiss Webster mice (26–30 g, Harlan Sprague Dawley, Inc.) were killed by cervical dislocation. The ileum was immediately dissected and placed in ice-cold Krebs solution (in mM: 118 NaCl, 4.6 KCl, 1.3 NaH2PO4, 1.2 MgSO4 25 NaHCO3, 11 glucose and 2.5 CaCl2) bubbled with carbogen (95% O2/5% CO2). Ileal segments were threaded longitudinally on a plastic rod through the lumen and the longitudinal muscle with the myenteric plexus (LMMP) was gently removed using a cotton-tipped applicator. LMMP strips were rinsed three times in 1 ml Krebs and gathered by centrifugation (350× g, 30 s). LMMP strips were then minced with scissors and digested in 1.3 mg/ml collagenase type II (Worthington) and 0.3 mg/ml bovine serum albumin in bubbled Krebs (37°C) for 1 hour, followed by 0.05% trypsin for 7 min. Following each digestion, cells were triturated and collected by centrifuge (350× g for 8 min). Cells were then plated on laminin (BD Biosciences) and poly-D-lysine coated coverslips in Neurobasal A media containing B-27 supplement, 1% fetal bovine serum, 10 ng/ml glial cell line-derived neurotrophic factor (GDNF, Neuromics, Edina, MN), and antibiotic/antimycotic liquid. Half of the cell media was changed every 2–3 days...

Amber Young, Kristin S. Assey, Carla D. Sturkie, Franklin D. West, David W. Machacek, Steven L. Stice. Glial cell line-derived neurotrophic factor enhances in vitro differentiation of mid-/hindbrain neural progenitor cells to dopaminergic-like. doi/10.1002/jnr.22499/abstract.

...At 24 hours post-plating, the medium was changed to neural differentiation medium (proliferation medium without bFGF) plus or minus 25 ng/ml GDNF (Neuromics)...