Monday, September 19, 2016

Your Neuron-Glia Co-Cultures Deserve the Best

There is a lot of cross talk between neurons and glia. In neuro-immuno and neuro-degenerative diseases this cross talk is dysregulated. Robust neuron-glia co-cultures are needed for drug discovery and basic research.Here is a study that references use of co-culturing to study the interactive effects of the viral protein HIV-1 Tat lipopolysaccharide (LPS) on enteric glia and neurons.

Here is a study that references use of co-culturing to study the interactive effects of the viral protein HIV-1 Tat lipopolysaccharide (LPS) on enteric glia and neurons. In order to optimize the co-culturing our Media, Coatings and GDNF growth factor are used. 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.

Figures: (A) Representative confocal microscopic images of enteric β-III tubulin (green) and glial fibrillary protein (GFAP- red) showing the presence of neurons and glia isolated from the adult mouse LMMP. (B) IL-6 release from isolated mouse neuron/glia co-culture treated with 100 nM Tat, 100 ng/ml LPS or Tat+ LPS for 16 h measured by ELISA. (C) IL-1β release from neuron/glia co-culture treated with 100 nM Tat, 100 ng/ml LPS and Tat+ LPS for 16 h measured by ELISA (D) mRNA expression of TNF-α of mouse LMMP treated with 100 nM Tat, 100 ng/ml LPS and Tat+ LPS for 16 h. (E) TNF-α release from mouse LMMP treated with 100 nM Tat, 100 ng/ml LPS or Tat+ LPS for 16 h measured by ELISA.

Protocol: 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.

We welcome your questions input on co-culturing. Pete Shuster, CEO and Owner , or 612-801-1007.

Tuesday, September 13, 2016

Perky ERKies and Depression

ERK1/2 Signaling and Depression

Extracellular signal-regulated kinase 1/2- (ERK1/2-) mediated cellular signaling plays a major role in synaptic and structural plasticity. Although ERK1/2 signaling has been shown to be involved in stress and depression. Phosphorylation of the receptor is important for maintaining healthy signaling, reduction of oxidative stress and induction of growth factors.

We are pleased that our ERK1/2 markers have been published in these type of studies. Here's a recent one: MARIA CONCETTA OLIANAS, Simona Dedoni, and PIER LUIGI ONALI. LPA1 mediates antidepressant-induced ERK1/2 signalling and protection from oxidative stress in glial cells. Published online before print September 7, 2016, doi: 10.1124/jpet.116.236455. ...Membranes were incubated overnight at 4 °C with one of the following primary antibodies: anti-phospho-ERK1 (Thr202/Tyr204) / ERK2(Thr185/Tyr187) (RA 15002) (1:15,000) (Neuromics, Northfield, MN, USA.

We will continue to post updates on this important topic

Sunday, September 04, 2016

Multiplexed Protein Array for Immunologists

Assay 1,631 Antibodies in Under 8 Hours!
The RayBio® Immunome Protein Arrays are multiplex protein immunoassays for numerous research applications. Correctly folded, functional proteins are immobilized onto a solid glass slide surface. The slides can then be probed with serum or small molecules and used in protein-antibody studies, small-molecule inhibition of kinase studies, protein-protein studies, methylation studies or DNA binding studies. Our main immunome array product offering contains 1,631 proteins. Smaller custom arrays are also available.

Each 75mm x 25mm glass slide is spotted with 4 identical protein arrays (also called “subarrays”), each with 1631 correctly folded and functional spotted proteins! Each protein is attached to the streptavidin-coated slide exclusively via a biotinylated affinity tag, specifically the biotin carboxyl carrier protein (BCCP) domain of the E. coli acetyl CoA carboxylase. This affinity tag was chosen because the BCCP domain must be folded into its native 3-dimensional structure in order to become biotinylated. If the expressed protein mis-folds the BCCP tag will also mis-fold, preventing biotinylation. If the protein folds correctly then folding of the BCCP tag should be unhindered and biotinylation will result. This property allows only correctly folded proteins to be bound to the slide.

Research Applications

  • Screening protein-antibody interactions 
  • Performing small-molecule-kinase inhibition experiments 
  • Screening protein-protein interactions 
  • Performing DNA-binding experiments Performing methylation assays
How it Works
Questions? Please contact me directly-Pete Shuster, CEO and Owner. 612-801-1007 or