Culturing Neurons on Conductive Biomaterials

Neuromics' Neurons on Graphene
Conductive biomaterials are an ideal bio-substrate for modifying cellular behaviors by conducting either internal or external electrical signals.

In this study, researchers successfully culture our cortical neurons on Nonfunctionalized graphene nanosheets (NGN): Shiyun Meng, Rong Peng. Growth and Follow-Up of Primary Cortical Neuron Cells on Nonfunctionalized Graphene Nanosheet Film. Article first published online: January 18, 2018. https://doi.org/10.5301/jabfm.5000263.

Figure: Spray-coating a nonfunctionalized graphene nanosheet (NGN) on a glass slide. To fix the NGN onto the glass slide, polyurethane (PU) was firstly spin-coated onto glass slides as a polymer matrix, then NGN particles were spray-coated on by airbrush.

Figure: Nuclei formed in 7 and 14 days of cell culture are shown at a relatively high magnification (scale bar = 50 μm): 1/500 MAP-2 (H-300) and 1/500 Alexa Fluor® 488 anti-Rabbit stained green for neuron microtubules and DAPI stained blue neuron nuclei.

We have a large catalog of potent, proven and pure human and animal cells.

Neurotrack Player, Essen Bioscience & Neuromics Neurons

I claim that our solutions are thoroughly tested, validated and research ready. We measure our performance by making sure this claim rings true with our customers.

One way this is confirmed to me is when a partner has success using one of our solutions in a new way. With that I am pleased to update you on Essen Bioscience's application for our primary neurons. Please note the related assays are being demonstrated in their most current webinar.

We will be featuring kinetic and migration assays using our hMSCs, hOsteoblasts and hChondrocytes in the near future. So stay tuned.

Primary Neuron Assays for Studying Neurodegeneration

Our goal is to provide our customers and collaborators the tools they need to insure success. This is defined by having the specific Primary Neurons, Growth Factor plus the Markers to meet unique research needs.

The proof is in the results. Here are some highlights.

Images/Data: FIGURE 5. Microglial p38α MAPK-dependent TNFα is involved in LPS-induced neurite degeneration. (A) Photomicrographs of MAP-2 immunocytochemistry show the morphology of neurons after 72h of co-culture with microglia. The arrow points to the appearance of neurites that have been damaged by LPS-activated WT microglia. In contrast, the arrowhead points to the morphological appearance of healthy, undamaged neurites. (B) Diagram of the Sholl method for quantifying the total number of healthy neurites that intersect the concentric circles. (C) Quantification of healthy neurites by the Sholl analysis demonstrates that LPS stimulation of p38α WT microglia in co-culture causes neurite degeneration as seen by a significant reduction in the number of intersections by healthy neurites in the LPS-stimulated group compared to the unstimulated group (white bars). This degeneration can be attenuated by the addition of a blocking antibody to TNFα (5μg/ml), while the non-immune IgG control was not protective (gray bars). Microglia from p38α KO mice stimulated with LPS (black bar) also have significantly less neurite degeneration than the LPS-stimulated p38α WT microglia (white bar). However, by adding TNFα back to the p38α KO microglia co-culture, there is a significant decrease in the healthy neurite arborization compared to the p38α KO microglia stimulated with LPS alone (black bars). (***p<0.005; Bonferroni’s multiple comparison test). Data represents 2 independent experiments. Scale bar equals 25μm. Molecular Neurodegeneration 2011, 6:84 doi:10.1186/1750-1326-6-84

hN2 cells grown in culture for 4 days and stained with our chicken polyclonal to Neurofilament light or low molecular weight chain NF-L, a marker of neurons. Many of the differentiating cells show strong cytoplasmic and clearly fibrillar staining for NF-L. Blue stain is DAPI and reveals cell nuclei of some non neuronal cells in this culture.

We will continue to post relevant images and data that demonstrate our capabilities.

Immunostaining Neurons and Glia

I would like to thank Dr. Gerry Shaw, University of Florida for his excellent work with our Primary Neurons and Astrocytes and Neuronal-Glial Markers. Here's an example image with many more to follow:

Image: E18 hippocampal neurons stained with MAPT (red) and Doublecortin (green). The two proteins overlap in the proximal dendrites, but doublecortin is more abundant in the growth cones and periphery. As a result, the periphery appears green while the more proximal regions of the cells are yellow. The single longer process of this cell, presumably an axon, has a low doublecortin content and so appears red. Blue staining is the nuclear DNA. Protocol on datasheet.

Xona Microfluidics and Neurons

I am impressed with these Video from the Jeon Lab at UC Irvine. It represents a novel method for neuro-drug discovery:
Preparing E18 Cortical Rat Neurons for Compartmentalization in a Microfluidic Device.

This technology represents a way to separate axon from cell bodies.

Primary Neurons and Cell Based Assays

The feedback I receive from Neuroscientists is consistent. To paraphrase, "gives us healthy, consistent and potent primary cells. I understand the hard work it takes to generate meaningful and publishable results from cell based assays. Our Primary Neurons and Astrocytes are merely inputs for these assays. The real cost is the time invested in culturing and time lost if they don't work.

I have numerous postings on success: Primary Neurons Postings. I wanted to share more data and feedback.

Primary DRGs-Culturing these can be tricky. I make it a point to work with labs to make sure the protocol options best match the desired outcome for assays. This includes replacing cells to make sure we can accurately troubleshoot. This approach insures I can pin point the issues and make sure they are all resolved in round two. Here's a representative testimonial: "Thanks for following up, the DRGs worked great and we were able to get excellent data from them. Thanks so much for working with us." Adam Ross, Dr. Chengji Zhou Lab, UC Davis

Image: DRGs cultured on Calf Skin Collagen.

Primary Hippocampal Neurons-I would like to thank Vimal Swarup, University of Utah for this excellent image.

The cells have been fixed after 48 hrs, they were grown over poly-lysine coated coverslips in the media supplied by Neuromics. Cells were imaged in phase contrast mode with 40x objective.

Put our primary cells to the test!

Primary Hypothalamic Neurons Cultures

Our customers have had great success dissociating and culturing our Fresh E18 and E20Rat Primary Neuronal Tissue. They have proven useful for a variety of cell based assays.

Here're several publications referencing use of the neurons:

Lisette T. Arnaud, Natura Myeku and Maria E. Figueiredo-Pereira. Proteasome–caspase–cathepsin sequence leading to tau pathology induced by prostaglandin J2 in neuronal cells. Journal of Neurochemistry. Volume 110 Issue 1, Pages 328 - 342 .

Karunya K. Kandimalla1, Olenych G. Scott, Smita Fulzele1, Michael W. Davidson, Joseph F. Poduslo. Mechanism of Neuronal versus Endothelial Cell Uptake of Alzheimer's Disease Amyloid β Protein. PLoS ONE 4(2): e4627. doi:10.1371/journal.pone.0004627.

The most challenging of our neurons to culture are from the microdissect E18 rat Hypothalamus. Big Kudos to Dr. Dan Ryder for his excellent job culturing them. He also generously shared these images: (A) 5X1 (B) 40X5 Brightfield.

Related Reagents:
STEMEZ(TM) hN2 Human Neurons Discovery Kit
Frozen Primary Rat Cortical Neurons
E18 Rat Primary Neuronal Tissue - ASTROCYTE CULTURES
Neuron/Glial Marker Antibodies
Neurotrophins-Neuron/Glial Marker Recombinant Proteins

Amyloid Beta and E18 Primary Hippocampal Neurons

We would like to feature an interesting application of our E18 Rat Primary Hippocampal Neurons.

Related Publication:

Karunya K. Kandimalla1, Olenych G. Scott, Smita Fulzele1, Michael W. Davidson, Joseph F. Poduslo. Mechanism of Neuronal versus Endothelial Cell Uptake of Alzheimer's Disease Amyloid β Protein. PLoS ONE 4(2): e4627. doi:10.1371/journal.pone.0004627.

... Rat primary hippocampal (RPH) neurons were isolated from the hippocampii of 18-day-old embryonic Sprague Dawley rat brains (Neuromics, Edina, MN). The hippocampii were dispersed using a fire polished Pasteur pipette and plated on poly-D-lysine (Sigma-Aldrich, St. Louis, MO) coated glass cover slips in B-27 neurobasal medium containing 0.5 mM glutamine and 25 µM glutamate (Invitrogen, Carlsbad, CA). The neuronal cells were grown under 5% CO2 in an incubator maintained at 37°C until differentiation...

Images: A–D: Uptake of fluorescein labeled Aβ40 (F-Aβ40) and Alexa Fluor® 633 labeled transferrin (AF633-Trf), clathrin-mediated endocytosis marker, in rat primary hippocampal (RPH) neurons following 30 min incubation at 37°C. (A) F-Aβ40 uptake; (B) Uptake of AF633-Trf; (C) Superimposition of images A and B; (D) Overlay of fluorescence images on the DIC image of RPH neurons. E–G: Uptake of F-Aβ40 and AF633-Trf in RPH neurons at 4°C. (E) Uptake of F-Aβ40; (F) No significant neuronal uptake of AF633-Trf at 4°C; (G) Superimposition of images D and E on the DIC image of RPH neurons; H–J: Uptake of F-Aβ40 and AF633-Trf in RPH neurons treated with 10 mM Sodium Azide and 50 mM 2-deoxy glucose, agents that are known to deplete cellular ATP. (H) Uptake of F-Aβ40; (I) No significant cellular uptake of AF633-Trf was observed; (J) Superimposition of images H and I on the DIC image of neurons.doi:10.1371/journal.pone.0004627.g010

Neurotoxicity Testing

We combine our expertise in providing fresh and healthy:

E18 and E20 Rat Primary Neuronal Tissue -NEURON CULTURES

E18 Rat Primary Neuronal Tissue - ASTROCYTE CULTURES

E18 Mouse Neuronal Tissue -NEURON CULTURES

E18 Mouse Neuronal Tissue -ASTROCYTE CULTURES

AND Apoptosis Research Reagents

to help researchers more effectively study Neurotoxicity.

Images: Polycaspase Assay Kit, green was used to assess cell death in primay rat hippocampal neurons.Cells were plated on 25-mm poly-l-lysine-coated coverslips at 300,000 cells per coverslip. Cells were used at 4 or 8 days in vitro. Composite imagae (A) 3 out of 4 cells are apoptotic (green). No cells were necrotic as both of the PI-positive cells were FLICA-positive; they had compromised membranes and were probably in the late stages of apoptosis rather than necrosis. (B) 3 Caspase-positive cells fluoresce green.

Combined Neuron Cultures Assays

We would like to thank Dr. Matteo Porotto, Weill Cornell Medical College

Larger Image

Primary rat neurons: Transfection of functional HeV glycoproteins and infection with HeV pseudotyped virions.In order to establish the feasibility of carrying out the proposed experiments in primary neurons, we show (figure ) that our assays are amenable to use in primary neurons. In the experiment, Combined Hippocampus, Cortex, and Ventricular -E18 (Neuromics) were plated, and at 3 days were transfected with plasmids encoding HeV G/F as well as YFP. On the following day, these cells were infected with HeV or VSV pseudotyped viruses bearing RFP. In the figure, (A) the phase contrast photos show the differentiated neurons; (B) upon excitation for RFP, the red fluorescence indicates neurons infected by HeV pseudotyped virions; (C) upon excitation for YFP, and the green fluorescence shows the efficiency of transfection in neurons. This experiment indicates that the proposed experiments can be carried out in primary neurons, which are transfectable and infectable in our systems, and thus supports all the proposed aims. Data Courtesy of Dr. Matteo Porotto, Weill Cornell Medical College

Primary Neuron Cultures Featured
Combined Hippocampus, Cortex, and Ventricular -E18
Other Reagents to Consider
All Primary Neurons/Astrocytes, Stem Cells and Media
Transfection Reagents
Neuron-Glial Markers
Stem Cell Markers

Recent Pubs Referencing Neuromic's Products

Neurotensin Receptor 1 (NTS1)-GP14020



Geneviève Roussy, Marc-André Dansereau, Louis Doré-Savard, Karine Belleville, Nicolas Beaudet, Elliott Richelson and Philippe Sarret. Spinal NTS1 receptors regulate nociceptive signaling in a rat formalin tonic pain model. doi:10.1111/j.1471-4159.2007.05205.x


Images: Expression of NTS1 receptors in sensory neurons and spinal cord. (a) Immunoperoxidase staining reveals the presence of NTS1-like immunoreactivity throughout the lumbar dorsal horn of the spinal cord. Immunolabeling is most prominent in the superficial layers of the dorsal horn. The lateral spinal nucleus (lsn) is also moderately labeled (arrow). (b) High magnification of (a). A dense NTS1 immunostaining is observed over laminae I and II of the dorsal horn. Numerous immunopositive nerve cell bodies are visible in the superficial laminae and in the nucleus proprius of the dorsal horn (arrows). (c) Light microscopic analysis of NTS1 expression in primary afferent neurons. NTS1 is expressed in subpopulations of small- and medium-ganglion cells. No apparent labeling is detected in large DRG neurons (arrows). At higher magnification, NTS1 neurons exhibit a cytoplasmic pattern of immunoreactivity (d, arrowheads). (e) Identification of endogenously expressed NTS1 receptors, by western blotting, in homogenates from DRGs, lumbar spinal cord and brain. The 47 kDa protein band corresponds to the molecular weight deduced from the cDNA sequence of NTS1. Each lane represents the transfer of 25 lg of protein. Scale bars: (a, b, c, and d) 300, 150, 70 and 20 lm, respectively. doi:10.1111/j.1471-4159.2007.05205.x

Primary Neurons and Astrocytes


Cortex
Hippocampus
Striatum
E18 and E20 Rat Primary Neuronal Tissue -NEURON CULTURES
E18 Rat Primary Neuronal Tissue - ASTROCYTE CULTURES
E18 Mouse Neuronal Tissue -NEURON CULTURES
E18 Mouse Neuronal Tissue -ASTROCYTE CULTURES
Frozen Primary Rat Neurons-NEW
Frozen Primary Mouse Neurons and Astrocytes-New
FluoGreen Tracer-Stains Cell Nuclei and Cytoplasm

Primary Neurons and Astrocytes

Shouqing Luo, Haruo Mizuta and David C. Rubinsztein. p21-activated kinase 1 promotes soluble mutant huntingtin self-interaction and enhances toxicity. Human Molecular Genetics 2008 17(6):895-905; doi:10.1093/hmg/ddm362

...Rat E18 cortical and striatal neurons were purchased from Neuromics (MN) or prepared from E18 rat brain tissues according to instructions from Neuromics, cultured in neurobasal (Invitrogen) medium supplemented with B27 (Invitrogen) and 0.5 mM glutamine and transfected with Lipofectamine 2000 according to standard methods...

Striatal Neurons Image

Thank you to Dr. Henry Guo, Intracellular Therapies

Larger Striatal Image (10X)

E18 and E20 Rat Primary Neuronal Tissue -NEURON CULTURES
Fresh Tissue and media are provided to initiate a culture of primary neurons.
E18 Rat Primary Neuronal Tissue - ASTROCYTE CULTURES
Fresh Tissue and media are provided to initiate a culture of primary astrocytes.
Human Brain Tissue (Blocks, Lysates and Slides)-New
Healthy adult human brain tissue
Frozen Primary Rat Neurons-NEW
From E18 Rat Cortex
Frozen Primary Mouse Neurons-New
Growth Media and Poly-D-Lysine Coverslips