Proud Distributor of RayBiotech's Antibody Arrays and ELISAs

Pioneer and Market Leader

I am pleased to announce we are a distribution partner for RayBiotech. The solutions we offer include Antibody Arrays and ELISA Kits. With these additions, you now have many options for the either the qualitative, semi-quantitative or quantitative measurement of protein expression. From single antibody pair ELISAs to multiplexed arrays. The arrays enable you to simultaneously analyze up to 80 cytokine, inflammatory response, growth factor or angiogenesis related proteins.

In this posting, I focus on our new C-Series Antibody Array Kits. The kits utilize the sandwich immunoassay principle, wherein a panel of capture antibodies is printed on a nitrocellulose membrane solid support (usualy 2.5 cm x 3 cm). The array membranes are processed similarly to a Western blot (chemiluminescent readout). Signals are then visualized on x-ray film or a digital image, allowing densitometry data collection and calculation of fold-changes for each detected protein. The entire procedure can be completed in 1 day, and is simple enough to permit even the novice researcher to successfully collect data with very few pitfalls and little or no optimization.

They are proven and frequently published. You can now analyze the protein expression in you assays for a fraction of the cost of tradition methods. Here's an example: Simona Giorgini, Daniela Trisciuoglio, Chiara Gabellini, Marianna Desideri, Laura Castellini, Cristina Colarossi, Uwe Zangemeister-Wittke, Gabriella Zupi and Donatella Del Bufalo. Modulation of bcl-xL in Tumor Cells Regulates Angiogenesis through CXCL8 Expression. doi: 10.1158/1541-7786.MCR-07-0088. Mol Cancer Res August 2007 5; 7

Figure: bcl-xL overexpression in ADF glioblastoma and M14 melanoma cells increases CXCL8 expression. Image of membranes from a protein array. Membranes are probed with CM from the (A) glioblastoma control (AN8) and bcl-xL–overexpressing (AXL42 and AXL74) clones and (B) melanoma control (Mneo) and bcl-xL–overexpressing (MXL12 and MXL90) clones. In the negative control (NEG), the CM is replaced with an appropriate mock buffer according to array protocol. The intensity of protein signals for each membrane was compared with the relative positive signals by densitometric analysis. C. Schematic representation of proangiogenic factors that can be detected by the use of the membrane. The CXCL8, TGF-1β, TIMP1, TIMP2, and VEGF protein signals (two spots) are indicated by red, blue, yellow, violet, and green rectangles, respectively, in each image....The Human Angiogenesis Antibody Array I (RayBiotech. Inc.) was used according to the manufacturer's protocol to evaluate the secretion of 20 angiogenic factors into the CM of the different lines. A schematic representation of the proangiogenic factors that may be detected by the use of the array has been reported in the above figure. Membranes spotted in duplicate with antibodies against angiogenic factors were blocked with blocking buffer and then were incubated overnight with CM. Next, membranes were washed with wash buffer, incubated with biotin-conjugated antibodies against proangiogenic factors, washed with wash buffer, and incubated with horseradish peroxidase–conjugated streptavidin. The signals on the membranes were detected by chemiluminescence. Membranes, blocking and wash buffer, and antibodies against proangiogenic factors were all provided with the kit. The intensity of protein signal (two spots for each protein) was compared with the relative positive signals by densitometric analysis.

Here's an excellent video that provides more detail on Antibody Array Kit capabililities:

I will continue to post information on new addition with related pubs and data.

A Method for 3D Immunostaining and Optical Imaging of the Mouse Brain

Demonstrated in Neural Progenitor Cells

We have proven and effective tools for delivering siRNA, shRNA and miRNA for gene expression analysis and measuring apoptosis in vivo.

We are pleased to introduce a new application for one of our neural progenitor markers. In this study our Chicken Nestin Antibody is used to stain these progenitors in dissected mouse brains: Gleave JA, Lerch JP, Henkelman RM, Nieman BJ (2013) A Method for 3D Immunostaining and Optical Imaging of the Mouse Brain Demonstrated in Neural Progenitor Cells. PLoS ONE 8(8): e72039. doi:10.1371/journal.pone.0072039. 3D antibody staining of adult mouse brains: 1% PFA perfused adult mouse brains were removed from the skull and then divided by removing the cerebellum and separating the hemispheres if desired. The samples were immediately dehydrated in a gradient of methanol solutions to 100% methanol over the course of one day. Immediate dehydration is important to preserve the cellular morphology of the lightly-fixed brain. The samples then underwent freeze/thaw (one hour at –80°C/one hour at room temperature) four times. The samples were rehydrated in a gradient of methanol solutions to PBS over the course of one day. The samples were mildly digested for 5 minutes using 10 mg/ml of Proteinase K (Promega, WI, USA) and then thoroughly washed to remove any residual Proteinase K. Antigen retrieval was performed using a Histos5 histology microwave (Milestone, MI, USA) in 0.01 M sodium citrate buffer pH 6 on an antigen retrieval program (0–90°C 11min, 90–98°C 3min, 98°C 10min). The samples were cooled to RT and subsequently washed in PBS to remove excess buffer. From this point, all incubations were performed in the Histos5 histology microwave. The samples were incubated at 37°C for 48 hours in primary antibody diluted in 5% normal goat serum (Jackson ImmunoResearch, PA, USA), 5% dimethylsulfoxide (DMSO, Fisher Scientific, Ontario, Canada), and 0.01% Triton X-100 (Bioshop Canada, Ontario, Canada). The samples were then washed at 37°C for 48 hours in 5% serum, 0.01% Triton X-100, replacing the solution once after 24 hours. Following this, the samples were incubated for 72 hours in secondary antibody diluted in 5% serum, 5% DMSO, and 0.01% Triton X-100. Finally, the samples were washed at 37°C for 48 hours in 0.01% Triton X-100, replacing the solution once after 24 hours.

Staining of single hemisphere brain samples using the described protocol for 3D imaging included the following: doublecortin (n = 4), GFAP (n = 4), nestin (n = 3), double stain of doublecortin and nestin (n = 1). Doublecortin was also used to stain a full mouse brain excluding the cerebellum (n = 2). Control samples were generated using pre-absorbed primary antibodies when possible by incubating the primary antibody with a blocking peptide for 30 minutes at room temperature prior to staining. If no peptide for the primary antibody was available, the staining procedure was performed in the control except that the primary antibody was omitted during the appropriate step.

Staining was performed with the following antibodies: rabbit anti-doublecortin (Abcam), goat anti-doublecortin and peptide (Santa Cruz Biotechnology) rabbit anti-GFAP (Dako), chicken anti-nestin (Neuromics), goat anti-rabbit alexafluor 546 (Invitrogen), goat anti-rabbit alexafluor 488 (Invitrogen), goat anti-rabbit alexafluor 647 (Invitrogen), goat anti-chicken alexafluor 546 (Invitrogen), goat anti-chicken alexafluor 488 (Invitrogen), donkey anti-goat alexafluor 546 (Invitrogen), donkey anti-goat alexafluor 488 (Invitrogen).

Images: Validation of 3D nestin staining. Cryosections cut from a stained brain hemisphere show nestin-positive cells (red; arrows, A). Some additional punctate fluorescence was present (arrowheads, A). The sections were re-probed with a secondary antibody (green; A) to highlight primary antibody left unbound by secondary antibody. No evidence of primary antibody left unbound by secondary antibody was found. Sections were also re-stained with both primary and secondary antibodies (B) to highlight primary-antibody antigen sites unoccupied. There is overlap of the original 3D nestin stain with the 2D re-stain (arrowheads). LV = Lateral ventricle, scalebars = 100 µm. doi:10.1371/journal.pone.0072039.g003

The best news of all: This staining method is simple, using a combination of heat, time and specimen preparation procedures readily available, so that it can be easily implemented without the need for specialized equipment, making it accessible to most laboratories.

Healthy and Happy Neuron/Astrocytes Cultures

A Track Record of Customer Success

Neuromics is  recognized for the quality of  hNP1™ Human Neural Progenitor,  hN2™ Neuron Discovery Kits, E18 and E20 Rat Primary Neurons and E18 Rat Primary Astroglia. As the company owner, it is important that I keep my finger on the pulse of how well they work for each and every unique application. I personally follow up with each user and if there are any issues, we replace the cells once free of charge. Your success is critical to our growth.

I wanted to share with you recent references. These give and excellent snapshot of the exciting ways our cells can used. Alexzander Asea, Punit Kaur, Alexander Panossian, Karl Georg Wikman, Evaluation of molecular chaperons Hsp72 and neuropeptide Y as characteristic markers of adaptogenic activity of plant extracts. Phytomedicine, Available online 6 August 2013, ISSN 0944-7113, http://dx.doi.org/10.1016/j.phymed.2013.07.001
...using trypan blue exclusion test and routinely found to contain less than ;5% dead cells. Primary human neurons were purchased from Neuromics (Edina, MN)...

Images: Micropictograph of primary culture from micro-dissected hippocampus. (A) Neurons are round and healthy 1 h after plating on poly-d-lysine substrate. (B) Five days in culture, neurons remain healthy and have extended processes. Magnification 60×. http://dx.doi.org/10.1016/j.phymed.2013.07.001

Todd GK, Boosalis CA, Burzycki AA, Steinman MQ, Hester LD, et al. (2013) Towards Neuronal Organoids: A Method for Long-Term Culturing of High-Density Hippocampal Neurons. PLoS ONE 8(4): e58996. doi:10.1371/journal.pone.0058996
... a protocol that allows for culturing of E18 hippocampal neurons at high densities for more than 120 days. These cultured hippocampal neurons are (i) well differentiated with high numbers of synapses, (ii) anchored securely to their substrate, (iii) have high levels of functional connectivity, and (iv) form dense multi-layered cellular networks. We propose that our culture methodology is likely to be effective for multiple neuronal subtypes–particularly those that can be grown in Neurobasal/B27 media. This methodology presents new avenues for long-term functional studies in neurons...
Xiugong Gao, Hsiuling Lin, Radharaman Ray, Prabhati Ray. Toxicogenomic Studies of Human Neural Cells Following Exposure to Organophosphorus Chemical Warfare Nerve Agent VX. Neurochemical Research. February 2013.
...Human hN2 neurons were obtained from Neuromics...

Image: Staining of hN2 Human Neurons with Tuj 1 (Neuron-specific class III beta-tubulin) (red) and Nestin (green). Counter stained with DAPI (blue). hN2 Cells-Electro Phys Data. 

Xiufang Guo, Severo Spradling, Maria Stancescu, Stephen Lambert, James J. Hickman. Derivation of sensory neurons and neural crest stem cells from human neural progenitor hNP1. Biomaterials, In Press, Corrected Proof,Mar 2013.doi:10.1016/j.biomaterials.2013.02.061
...hNP1, were obtained from Neuromics (Edina, Minnesota)...
Wei Zhang , Radhia Benmohamed, Anthony C. Arvanites, Richard I. Morimoto, Robert J. Ferrante, Donald R. Kirsch, Richard B. Silverman. Cyclohexane 1,3-diones and their inhibition of mutant SOD1-dependent protein aggregation and toxicity in PC12 cells. Bioorganic & Medicinal Chemistry. Elsevier Ltd. All rights reserved.doi:10.1016/j.bmc.2011.11.039.
...Primary rat cortical tissue was purchased from Neuromics Inc., Edina, MN and used to initiate primary cortical neuron cultures. The tissue was isolated from micro-surgically dissected E18 embryonic Sprague/Dawley or Fischer 344 rat brain and shipped in a nutrient rich medium under refrigeration. To isolate neurons, the tissue was incubated with papain at a concentration of 2 mg/mL in Hibernate without calcium for 30 min at 37^OC. The enzymatic solution was then removed, and 1 mL of culture media (Neurobasal, B27, 0.5 mM glutamine) was added. A sterile Pasteur pipette was used to gently disperse the cells, which were then washed, re-suspended and counted. The cells were plated on poly-D-lysine coated 96-well plates at a density of 20,000 cells/well and incubated at 37^OC in a 5% CO2-humidified atmosphere for 5 days prior to use in compound testing. By microscopic inspection, the resulting cultures consisted of app. 90% neurons...
Majumder A, Dhara SK, Swetenburg R, Mithani M, Cao K, Medrzycki M, Fan Y, Stice SL. Inhibition of DNA methyltransferases and histone deacetylases induces astrocytic differentiation of neural progenitors. Stem Cell Res. 2013 Jul;11(1):574-86. doi: 10.1016/j.scr.2013.03.003. Epub 2013 Apr 2.
...Progenitor to Astrocytes Protocol: For astrocytic differentiation of hNP cells, neuronal differentiation media were supplemented with BMP2 (20 ng/mL) and combinations of Aza-C and TSA; Aza-C (500 nM), TSA (100 nM) and BMP2 (20 ng/mL) for 2 days, with one complete media change in between, followed by differentiation media supplemented with BMP2 but not with Aza-C or TSA. Cells were harvested prior to analysis at 5, 15 or 30 days of treatment or for cryopreservation at d6 or d10 of differentiation. For cryopreservation, cells were dissociated with Accutase™ and frozen in differentiation media containing10% DMSO. Viability was assessed at 30 days in Aza-C and TSA treated cultures by trypan blue exclusion, and datawas acquired using a Cellometer Auto T4® (Nexcelom Biosciences)...
Aparna Talekar, Antonello Pessi, and Matteo Porotto. Infection of primary neurons mediated by Nipah virus envelope proteins: Role of host target cells in antiviral action. J. Virol. doi:10.1128/JVI.00452-11.
...Hippocampus, Cortex and Ventricular Cells (Neuromics)...

I will continue posting results here.

eSC Derived hNP1 Neural Progenitors Astrocytic Differentiation

Protocol for Driving hNP1^TM Human Neural Progenitors to Astrocytes

There is a great demand for an easy way to generate human astrocytes in culture. I am pleased to present a protocol for differentiating our hNP1 Cells to Astrocytes. This comes from my friend Dr. Steve Stice and his team at ArunA Biomedical and University of Georgia: Majumder A, Dhara SK, Swetenburg R, Mithani M, Cao K, Medrzycki M, Fan Y, Stice SL. Inhibition of DNA methyltransferases and histone deacetylases induces astrocytic differentiation of neural progenitors. Stem Cell Res. 2013 Jul;11(1):574-86. doi: 10.1016/j.scr.2013.03.003. Epub 2013 Apr 2.

These enriched non-transformed human astrocyte progenitors will provide a critical cell source to further our understanding of how astrocytes play a pivotal role in neural function and development. Human neural progenitors derived from pluripotent embryonic stem cells and propagated in adherent serum-free cultures provide a fate restricted renewable source for quick production of neural cells; however, such cells are highly refractive to astrocytogenesis and show a strong neurogenic bias, similar to neural progenitors from the early embryonic central nervous system (CNS). We found that several astrocytic genes are hypermethylated in such progenitors potentially preventing generation of astrocytes and leading to the proneuronal fate of these progenitors. However, epigenetic modification by Azacytidine (Aza-C) and Trichostatin A (TSA), with concomitant signaling from BMP2 and LIF in neural progenitor cultures shifts this bias, leading to expression of astrocytic markers as early as 5days of differentiation, with near complete suppression of neuronal differentiation.

Images: Morphology and gene expression after 15 and 30 days of differentiation of cells with astrocytic treatment. Bright field images of hNP cells differentiated (A) with or (B) without astrocytic treatment. A and B compare morphology of cultured cells in treated vs. untreated differentiation at 15 days. Treated and untreated cells were cryopreserved at d6 and subsequently thawed and cultured for an additional 9 days. Flow cytometry analysis to determine percent of GFAP+ and S100B+ cells at d15 of differentiation. Data is presented as histograms for (C) GFAP and (D) S100B with corresponding immunoreactive cells in insets from a parallel culture. Immunocytochemistry detects expression of (E) GFAP with S100B (inset showing distinct staining for both markers), (F) GFAP with GLAST, and (G) GFAP with ALDH1L1 at d30 of differentiation.

The Protocol:  For astrocytic differentiation of hNP cells, neuronal differentiation media were supplemented with BMP2 (20 ng/mL) and combinations of Aza-C and TSA; Aza-C (500 nM), TSA (100 nM) and BMP2 (20 ng/mL) for 2 days, with one complete media change in between, followed by differentiation media supplemented with BMP2 but not with Aza-C or TSA. Cells were harvested prior to analysis at 5, 15 or 30 days of treatment or for cryopreservation at d6 or d10 of differentiation. For cryopreservation, cells were dissociated with Accutase™ and frozen in differentiation media containing10% DMSO. Viability was assessed at 30 days in Aza-C and TSA treated cultures by trypan blue exclusion, and datawas acquired using a Cellometer Auto T4® (Nexcelom Biosciences).

I will keep you updated on new differentiation protocols for our potent, pure and widely used hNP1 Human Neural Progenitors to new phenotypes.

MAP-2-A Versatile Neuron Marker

Neuromics is a leader in providing Neuron-Glial Markers for Neuroscientists.

We are constantly on the search for publications that reference use of these markers in unique applications. In this posting I would like to share a publication where researchers used on of our MAP-2 antibodies to stain medial superior olive (MSO) neurons. Baumann Veronika, Lehnert Simon, Leibold Christian, Koch Ursula. Tonotopic Organization of the Hyperpolarization-activated Current (Ih) in the Mammalian Medial Superior Olive. Front. Neural Circuits 7:117. doi: 10.3389/fncir.2013.00117.
 ...Following recording, slices were fixed in 4% paraformaldehyde for 30 min. After extensive washing in phosphate-buffered saline (PBS) slices were exposed to blocking buffer (0.5% trition X-100/0.1% saponin/1% BSA in PBS) followed by incubation with the primary antibody (chicken anti-microtubule-associated protein 2, MAP2, 1:1000, Neuromics) in blocking buffer. Slices were then rinsed in washing buffer (0.5% Trition X-100/0.1% saponin in PBS) and immunoreactivity was visualized by incubating the slices with the Cy3-conjugated secondary antibody raised in donkey (1:300; Dianova). Finally, slices were washed and mounted on slides with vectashield mounting reagent (Vector Laboratories, USA)...

Here the MAP-2 antibody is used to help identify the dorsal, medial and ventral portion of the MSO of p18 and p22 gerbils.

Figure . Ih varies systematically along the dorsoventral axis. (A) A brain slice containing the MSO with Alexa-488-filled neurons (green) verifies the distribution of the patched neurons along the dorsoventral axis (red: MAP-2). (B) Pharmacologically isolated Ih current traces were elicited by depolarizing and hyperpolarizing voltage steps from −60.5 mV to potentials between −40.5 mV and −120.5 mV for 1 s in 5 mV step increment and then to −100.5 mV for 0.5 s to elicit the tail current to determine the voltage dependence of Ih activation. Current traces are representative for the dorsal, the intermediate and the ventral part of the MSO. (C) I-V relationships of steady-state (red arrow in B) Ih density for ventral (n = 15), intermediate (n = 12) and dorsal (n = 18) neurons emphasize that Ih density amplitudes are smallest in dorsal neurons and largest in ventral neurons (C1). Ih density amplitudes for a voltage step to −110.5 mV (C2). (D) Weighted activation time constants at −110.5 mV (D1). The weighted activation time constants are voltage dependent and largest in the dorsal part of the MSO (D2). (E) The voltage-dependence of Ih activation was measured from the tail current 20 ms after the end of the voltage steps (red arrow) (E1). Values were fitted with a Boltzmann function to obtain the half-maximal activation voltage. In dorsal neurons the Ih activation curve is shifted to more negative voltages (E2). Half-maximal activation voltage was measured in each experiment and averaged (E3). Black symbols: dorsal neurons; gray symbols: intermediate neurons; white symbols: ventral neurons. **P < 0.01, ***P < 0.001, single-factor ANOVA test followed by a Scheffe's post-hoc test.

I will continue to post interesting applications using our Neuron-Glial Markers.

Using hESCs to Understand Vasculogenesis Processes

The Role of BMP4 Plays in Regulating Vascular Development

My friend, Dr. Steve Stice and team at UGA have published excellent findings on the process of vasculogenesis: N. L. Boyd, S. K. Dhara, R. Rekaya, E. A. Godbey, K. Hasneen, R. R. Rao, F. D. West III, B. A. Gerwe, S. L. Stice. BMP4 Promotes Formation of Primitive Vascular Networks in Human Embryonic Stem Cell–Derived Embryoid Bodies. Exp Biol Med (Maywood) June 2007 vol. 232 no. 6 833-843.
Highlights:
Human embryonic stem cells (hESC) have the capability to produce all of the cells of the body and have been used as in vitro models to study the molecular signals controlling differentiation and vessel assembly. One such regulatory molecule is bone morphogenetic protein-4 (BMP4), which is required for mesoderm formation and vascular/hematopoietic specification in several species. However, hESC grown in feeder-free conditions and treated with BMP4 differentiate into a cellular phenotype highly expressing a trophoblast gene profile. Therefore, it is unclear what role, if any, BMP4 plays in regulating vascular development in hESC. Here we show in two National Institutes of Health–registered hESC lines (BG02 and WA09) cultured on a 3D substrate of Matrigel in endothelial cell growth medium–2 that the addition of BMP4 (100 ng/ml) for 3 days significantly increases the formation and outgrowth of a network of cells reminiscent of capillary-like structures formed by mature endothelial cells (P < 0.05). Analysis of the expression of 45 genes by quantitative real time–polymerase chain reaction on a low-density array of the entire culture indicates a rapid and significant downregulation of pluripotent and most ectodermal markers with a general upregulation of endoderm, mesoderm, and endothelial markers. Of the genes assayed, BMPR2 and RUNX1 were differentially affected by exposure to BMP4 in both cell lines. Immunocytochemistry indicates the morphological structures formed were negative for the mature endothelial markers CD31 and CD146 as well as the neural marker SOX2, yet positive for the early vascular markers of endothelium (KDR, NESTIN) and smooth muscle cells (α-smooth muscle actin [αSMA]). Together, these data suggest BMP4 can enhance the formation and outgrowth of an immature vascular system.

Figures: Least-squared mean gene expression analysis of BMP4 versus control time course for BGO2 and WA09. BG02 (solid lines) and WA09 (dashed lines) were cultured for 3 days ± BMP4, then for another 5 days in EGM-2 only. Samples were collected on Days 0, 2, 4, 6, and 8; total RNA was extracted, and gene expression was analyzed by qRT-PCR. Least-squared mean comparison was then expressed as x-fold change with respect to Day 0 control. The gene expression time course for (A) OCT4, (B) HEY1, (C) GATA3, (D) NESTIN, (E) SOX2, (F) BMPR2, (G) KDR/Flk1, and (H) CD31/PECAM1 are shown. (BG02: −BMP4 = solid diamond; +BMP4 = solid square; WA09: −BMP4 = open triangle; +BMP4 = open circle; * P < 0.05 for BG02 only; ** P < 0.05 for WA09 only; *** P < 0.05 for both BG02 and WA09).http://ebm.sagepub.com/content/232/6/833.full

Images: Early vascular markers KDR, αSMA, and NESTIN are detected within the network structures and elsewhere. Cell cultures were dual-immunostained to detect co-expression of KDR with αSMA (A–C), KDR with NESTIN (D–F), or αSMA with NESTIN (G–I), and the nucleus was counterstained with DAPI. For each case, three different morphologic regions were represented: the residual EB (A, D, and G), the periphery of the EB (B, E, and H), and the thin network structures (C, F, and I). All images were acquired with a ×40 oil objective, and a Z series stack was projected into a single image. A color figure is available in the online version of the journal.

These findings confirm the use of stem cells and the EB as a model of early development including vasculogenesis (3, 4). Using hESC in this manner can provide insights into the mechanisms regulating the earliest events in human vasculogenesis. An understanding of how the vasculature is formed could also be applied to tissue engineering and angiogenic/ischemic therapies. Note: our Mouse Monoclonal Nestin Antibody is an excellent marker for early vasculogensis of the endothelium.

Protocol for Flow Cytometric Sorting of Enriched Neuronal Cultures from iPSCs

Surface molecule profiles undergo dynamic changes in physiology and pathology, serve as markers of cellular state and phenotype and can be exploited for cell selection strategies and diagnostics. The isolation of well-defined cell subsets is needed for in vivo and in vitro applications in stem cell biology. In this technical report, the authors present an approach for defining a subset of interest in a mixed cell population by flow cytometric detection of intracellular antigens. They have developed a fully validated protocol that enables the co-detection of cluster of differentiation (CD) surface antigens on fixed, permeabilized neural cell populations defined by intracellular staining. Determining the degree of co-expression of surface marker candidates with intracellular target population markers (nestin, MAP2, doublecortin, TUJ1) on neuroblastoma cell lines (SH-SY5Y, BE(2)-M17) yielded a combinatorial CD49f^-/CD200^high surface marker panel. Its application in fluorescence-activated cell sorting (FACS) generated enriched neuronal cultures from differentiated cell suspensions derived from human induced pluripotent stem cells. Our data underlines the feasibility of using the described co-labeling protocol and co-expression analysis for quantitative assays in mammalian neurobiology and for screening approaches to identify much needed surface markers in stem cell biology: Turaç G, Hindley CJ, Thomas R, Davis JA, Deleidi M, et al. (2013) Combined Flow Cytometric Analysis of Surface and Intracellular Antigens Reveals Surface Molecule Markers of Human Neuropoiesis. PLoS ONE 8(6): e68519. doi:10.1371/journal.pone.0068519

Figure 1. Experimental outline. Schematic illustrating the research strategy of identifying novel surface marker combinations on a target population in neural and other stem cell differentiation systems for which intracellular, standard immunocytochemical markers are well established. Following harvesting, the resulting single cell suspension is subject to surface antigen candidate staining, followed by gentle fixation, permeabilization and subsequent co-staining with known intracellular markers. CD markers co-labeling the target population serve as positive markers, those absent on the target population serve as negative markers. In a separate, subsequent step, a combination of the identified positive and/or negative CD markers enables the flow cytometric enrichment of the viable population of interest from a heterogeneous cell suspension for further study and biomedical applications. doi:10.1371/journal.pone.0068519.g001.

Figure 2. Accurate detection of intracellular antigens with optimized fixation-permeabilization conditions preserving surface antigens. Flow cytometric detection of TUJ1, MAP2 and nestin antigens in BJ fibroblasts and the neural SH-SY5Y cell line (A). TUJ1 and nestin are present in both cell lines, while the mature neuronal marker MAP2 was only detected in SH-SY5Y cells (arrows). Note stable fluorescent levels of the negative population, indicating low background staining using this protocol. Representative experiment of three independent repeats shown. (B) Corresponding validation by immunofluorescence analysis. (C) Quantitation of TUJ1, MAP2 and nestin intracellular antigen detection (n=3). Error bars indicate standard deviation. (D) Response of TUJ1 and MAP2 intracellular antigen expression to 6 DIV of 10 µM retinoic acid (RA) treatment of SH-SY5Y cells. Note disappearance/reduction of subsets negative for these markers (upward shift, green arrows), as well as a shift toward CD184low expression with differentiation (blue arrows). doi:10.1371/journal.pone.0068519.g003.

Please note: In addition to the our Human Mouse Monoclonal Nestin Antibody used in the study, we have an extensive catalog of stem cell solutions. I will continue to post highlights of new applications.

Super-Charging Pluripotent Stem Cells

Adapting Stem Cells to Cellular Stress for Regenerative Medicine and Cell-Based Therapies

This approach for the isolation and characterization of a new population of adipose tissue (AT) derived pluripotent stem cells could represent a breakthrough. The authors have identified these cells as "Multilineage Differentiating Stress-Enduring (MUSE) Cells": Heneidi S, Simerman AA, Keller E, Singh P, Li X, et al. (2013) Awakened by Cellular Stress: Isolation and Characterization of a Novel Population of Pluripotent Stem Cells Derived from Human Adipose Tissue. PLoS ONE 8(6): e64752. doi:10.1371/journal.pone.0064752.

Highlights: Although adult stem cells have been considered an attractive source for cell therapy, their effectiveness and efficiency is hindered by a frequently low survival rate due to their exposure to a high cellular stress environment upon transplantation. This key limitation is observed when utilizing adult stem cells for regenerative purposes, as typical cell engraftment yields are extremely low (less than 3%). This low survival rate limiting in the use of stem cells for therapies.

The authors have developed methods for isolating MUSE Cells that are preconditioned to survive engraftments. These cells display down regulation of genes involved in cell death and survival, embryonic development, organism survival, cellular assembly and organization, mitosis, DNA replication, recombination and repair.

Figure 1. Isolation and morphologic characterization of Muse-ATs. (A) Schematic of Muse-AT isolation and activation from their quiescent state by exposure to cellular stress. Muse-AT cells were obtained after 16 hours, with incubation with collagenase in DMEM medium without FCS at 4°C under very low O2 (See Methods). (B) FACS analysis demonstrates that 90% of isolated cells are both SSEA3 and CD105 positive. (C) Muse-AT cells can grow in suspension, forming spheres or cell clusters as well as individual cells (see red arrows) or (D) Muse-AT cells can adhere to the dish and form cell aggregates. Under both conditions, individual Muse-AT cells reached a diameter of approximately 10µm and cell clusters reached a diameter of up to 50µm, correlating to stem cell proliferative size capacity. doi:10.1371/journal.pone.0064752.g001.

Muse-AT cell isolation requires a simple yet highly efficient purification technique, Muse-AT cells could provide an ideal source of pluripotent-like stem cells with the potential to have a critical impact on regenerative medicine and cell-based therapy. The capabilities of these cells need to be further validate. I will keep you posted.

mGluR5 and Fragile-X Syndrome (FXS)

Upregulation of mGluR5 uncovered in postmortem prefrontal cortex of 14 FXS patients

We are proud that our mGluR5 antibody was selected for this important study. The investigators found that mGluR5 binding density and protein expression were increased in the brains of FXS patients or carriers: Talakad G Lohith, Emily K Osterweil, Masahiro Fujita, Kimberly J Jenko, Mark F Bear, Robert B Innis. Is metabotropic glutamate receptor 5 upregulated in prefrontal cortex in fragile X syndrome? Molecular Autism 2013, 4:15 (24 May 2013).

Protocol: Tissue samples were homogenized using a mortar and pestle in fresh ice-cold, 50 mM Tris– HCl buffer (1:10 w/v) completing 3 × 10 passes with cooling on ice between homogenizations. Homogenates were centrifuged at 20,000g and 4°C for 25 minutes,followed by removal of the supernatant. Pellets were then resuspended in fresh ice-cold 50 mM Tris–HCl buffer and centrifuged again at the same settings. Pellets were resuspended in fresh ice-cold 50 mM Tris–HCl buffer at a protein concentration of approximately 1 mg of protein/mL. Aliquots were stored in a freezer at −80°C until further use. Protein concentrations were determined using the Bradford protein assay (Bio-Rad, Hercules, CA), and absorption was read at 595 nm.

Figure: mGluR5 receptor density and expression in prefrontal cortex of FXS individuals and healthy controls. (A) Binding curves from homologous competition binding of 0.48 nM of [3H] MPEPy to membrane preparation from FXS and control subject samples at concentrations of unlabeled MPEPy ranging from 0.1 nM to 1μM. Individual binding curves were obtained from the average of triplicate measurements for e ach unlabeled ligand concentration. Data represent mean ± standard error in the mean from 14 FXS patients or carriers (FX) and 17 healthy controls (HC). At low concentration of unlabeled ligand, specific binding was higher for FXS than control samples. (B) Results of unpaired t-test tocompare the two groups. mGluR5 density tended to be higher (+16%;P = 0.058) in FXS patients than in the control group. Data represent mean ± standard deviation. Solid triangles (▲) in the FX group indicate the location of three FXS carriers; semisolid triang les indicate the location of a carrier with FXTAS. (C) Representative immunoblot for mGluR5. The mGluR5 band intensity was stronger for the FXS than control subject. Total protein stain of the same lanes confirmed equal-protein loading. (D) Average mGluR5: total protein ratio normalized to control subjects. The ratio was high and marginally significant (+32%; P=0.048) for the FXS group compared with controls. Data represent mean ± standard deviation.Solid triangles (▲) in the FX group indicate the location of three FXS carriers; semisolid triangles indicate the location of a carrier with FXTAS.
Conclusions:
This study was the first to identify upregulation of mGluR5 density and expression in the prefrontal cortex of FXS patients or carriers compared to an age- and sex-matched control group. This is consistent with several studies in FXS model mice that postulate that the syndromic features of FXS are caused by an upregulated mGluR5 signaling pathway. Although the sample size was relatively small and the results could be secondary to prior medication treatment, these initial findings provide strong rationale for measuring mGluR5 in live patients using PET. Such in-vivo studies could measure mGluR5 in all brain regions; the results could also be correlated with treatment response to mGluR5 negative allosteric modulators.

Quantibody® Cytokine Arrays

Cytokines, Adipokines, Growth Factors and Neurotrophins

We are pleased to announce addition of Antibody Arrays to our catalog. These are excellent tools for study apoptosis, inflammation, angiogenesis and  immune response and related autoimmune, inflammatory and degenerative diseases plus many forms of cancers. Check out this video.

Specific Research Applications Include:
•High-throughput profiling of cytokine expression
•Validation of semi-quantitative antibody array results
•Identifying potential molecular targets for drug development
•Identifying the molecular mechanisms of drug action
•Identifying crucial factors involved in disease processes
•Discovering biomarkers for disease management
•Discovering expression patterns for molecular classification of diseases

How Quantibody Arrays Work

We will be aggresively adding new arrays to our product offerings in June. Stay tuned for more.





Schwann Cell-Sensory Neurons-PNS Markers

Data-Publications
These markers a important tools for the study of Neuro-muscular diseases like Amyotrophic Lateral Sclerosis (ALS) and Multiple Sclerosis (MS).

For Neuromuscular Disease Researchers, we have some of the best Schwann Cell and Sensory/Peripheral Neuron Markers in the business.

Images: Rat mixed neuron/glial cultures stained with Peripherin (green channel) and Neurofilament alpha-internexin/NF66 (green channel). These cultures contain mostly neurons which are rich in alpha-internexin, and a subgroup which have a large amount of peripherin also, such as the prominent cell in the middle of the micrograph. Since this cell expresses large amounts of peripherin and alpha-internexin, the green and red signals superimpose to produce a golden cell. Blue is a DNA stain. Protocol on data-sheet.

Here're recent publications referencing use of these markers:
Leah R. Reznikov, Qian Dong, Jeng-Haur Chena, Thomas O. Moninger, Jung Min Park, Yuzhou Zhang, Jianyang Du, Michael S. Hildebrand, Richard J. H. Smith, Christoph O. Randak, David A. Stoltz, and Michael J. Welsh. CFTR-deficient pigs display peripheral nervous system defects at birth. www.pnas.org/cgi/doi/10.1073/pnas.1222729110...goat anti-p75 (1:500; Neuromics)...

Gayle M. Passmore, Joanne M. Reilly, Matthew Thakur, Vanessa N. Keasberry, Stephen J. Marsh, Anthony H. Dickenson and David A. Brown. Functional significance of M-type potassium channels in nociceptive cutaneous sensory endings. Fronteirs in Molecular Science. doi: 10.3389/fnmol.2012.00063. ...neurofilament H (1:1000,Neuromics,USA)...

Leigh A Nattkemper, Zhong-Qiu Zhao, Anna J Nichols, Alexandru D P Papoiu, Carol A Shively, Zhou-Feng Chen and Gil Yosipovitch. Over-Expression of the Gastrin-Releasing Peptide in Cutaneous Nerve Fibers and its Receptor in Spinal Cord in Primates with Chronic Itch. Journal of Investigative Dermatology accepted article preview 4 April 2013; doi: 10.1038/jid.2013.166...Protein Gene Product 9.5 (PGP9.5; Neuromics, Edina, MN).

Wiebke Kallenborn-Gerhardt, Katrin Schröder, Domenico Del Turco, Ruirui Lu, Katharina Kynast, Judith Kosowski, Ellen Niederberger, Ajay M. Shah, Ralf P. Brandes, Gerd Geisslinger, and Achim Schmidtko. NADPH Oxidase-4 Maintains Neuropathic Pain after Peripheral Nerve Injury. The Journal of Neuroscience, 25 July 2012, 32(30): 10136-10145; doi: 10.1523/​JNEUROSCI.6227-11.2012...chicken anti-P-Zero or MPZ (1:500; Neuromics)...

Images: Micrographs depicting SSeCKS colocalization with myelination markers (CNPase and Pzero). (A) SSeCKS (red) and CNPase (green) in the lumbar spinal cord dorsal horn. The labeling appears discrete with minimal colocalization. (B) SSeCKS (red) and Pzero (green) in the L4 dorsal root ganglia. A lack of co-localization is observed and Pzero can be seen localized to putative axonal elements (arrow). (C) SSeCKS (red) and Pzero (green) in the sciatic nerve. As in the dorsal root ganglia, a lack of co-localization is observed. Both SSeCKS and Pzero can be seen localized to axonal elements. (D) SSeCKS (red) and Pzero (green) in glabrous skin of the hind-paw, fibers displaying colocalization (yellow) can be observed (arrow). Irmen et al. Journal of Brachial Plexus and Peripheral Nerve Injury 2008 3:8 doi:10.1186/1749-7221-3-8.

I will post new data and pubs as they become available.

The Role of Obesity in Obstructive Sleep Apnea (OSA)

Intermittent Hypoxia and Leptins

Scott Mesenger recently published a Master's Thesis that implicates compelling links between Leptin Signaling and OSA: http://ir.lib.uwo.ca/cgi/viewcontent.cgi?article=2487&context=etd. This work was done in the John Cirello Lab at University of Western Ontario. The study extensively uses our Ob-Rb Antibody to generate data.

There is a strong link between obesity and OSA. "Perhaps the single most important factor affecting OSA risk is body weight. Weight gain of 10% increases the risk of developing OSA by six-fold (Peppard et al., 2000a) and a 10% loss of body weight is estimated to decrease the apnea/hypopnea index (events/hour) by 26%  Within the obese population (BMI ≥ 30) (Wolk et al., 2003), approximately 40% experience significant OSA and approximately 70% of OSA patients are obese (Vgontzas et al., 1994), daunting numbers considering the obesity epidemic occurring in North America and estimates suggesting 41% of Americans will be obese by 2015 (Nejat et al., 2009)."

"Leptin can exert cardiovascular effects by acting centrally, as has been shown by studies introducing leptin directly into the CNS. Central administration serves to increase plasma catecholamines epinephrine and norepinephrine (Satoh et al., 1999). In other studies, leptin has been found to increase SNA, arterial pressure as well as heart rate and to inhibit the baroreflex (Arnold et al., 2009; Mark et al., 2009). Leptin may also have a role in modulating the peripheral chemoreflex at the level of the CNS as microinjection of leptin into caudal pressor areas of the NTS has been found to potentiate the sympathetic and blood pressure responses to chemoreflex activation (Ciriello & Moreau, 2012). These cardiovascular effects may not be debilitating under normal homeostatic conditions, however in conditions of increased circulating leptin, such as in OSA and obesity, the cardiovascular responses may be detrimental."

This study shows the increase in the expression of leptins like Ob-Rb in Carotid .

Images: Fluorescent (a-c) photomicrographs showing the effect of IH on Ob-Rb (a) and ERK 1/2 (b) expression in carotid body glomus cells. Note that Ob-Rb and ERK 1/2 are co-expressed in the same cells (c). Calibration mark in (a) represents 100 μm and applies to (a-c).

Images: Western blots showing the presence of Ob-Rb (a) and Ob-R100 (b) in carotid bodies after IH. Note that IH significantly (*) decreases the protein level of Ob-Rb (a), while significantly (*) increasing the protein level of Ob-R100 (b) compared to normoxic controls (b). p values are indicated. n=5-6.

This study will give the reader great insights on the molecular biology of OSA. Check it out: http://ir.lib.uwo.ca/cgi/viewcontent.cgi?article=2487&context=etd.

Tissue Acidosis and Inflammation Related Nociceptive Pain

Important Implications for Inflammatory Pain States

Inflammatory pain is often accompanied by a drop in pH (Acidosis). Here investigators hypothesized that modest drops in extracellular pH leading to calcium fluxes acts like a dynamic switch to rapidly mobilize trkA to the cell membrane surface of adult sensory neurons, which in turn serves to increase the sensitivity of these neurons to NGF. The findings reveal a cellular mechanism whereby even small changes in pH can rapidly shift sensitivity to a critical driver of the inflammatory pain state—NGF. They way they demonstrate this shift is quite ingenious: Geoffrey E. Bray, Zhengxin Ying, Landon D. Baillie, Ruiling Zhai, Sean J. Mulligan, and Valerie M.K. Verge. Extracellular pH and Neuronal Depolarization Serve as Dynamic Switches to Rapidly Mobilize trkA to the Membrane of Adult Sensory Neurons. The Journal of Neuroscience, 8 May 2013, 33(19):8202-8215; doi:10.1523/JNEUROSCI.4408-12.2013.

Images: NGF-responsive adult sensory neurons have a large cytoplasmic pool of high-affinity NGF receptors. Six micrometer cryostat sections of L5 DRG processed for high-affinity NGF binding (left) or trkA immunohistochemistry (right) reveal a large cytoplasmic pool of proteins that are able to bind 20 pm radio-iodinated NGF with high affinity or that are immunoreactive to trkA-selective antibodies. Scale bar, 20 μm.

Here're images showing the pH Related switch and rapid migration of TrkA to the membrane

Images: Acidic pH challenge rapidly mobilizes trkA to the membrane from internal stores. A, Bar graphs summarize relative changes in neuronal cell-surface trkA expression from three separate experiments (detected with immunofluorescence) over neurons exposed to control (pH 7.4) or acidic (pH 6.5) media for 30 min with or without exposure to Golgi collapsing compound BFA and as normalized to the mean signal intensity from the control pH group. Note: A significant increase is observed in the mean levels of trkA mobilized to the neuronal membranes of sensory neurons in response to acidosis when compared with the control pH, a response that is blocked with BFA treatment. B, Immunofluorescence photomicrographs and summary histograms (C) depict degree of FM 1–43FX-stained neuronal membrane internalization in response to conditions as indicated. Note: Significant membrane internalization was only observed in the NGF challenge control group and not in response to acidic pH challenge with or without BFA treatment. (Data normalized to the control pH of each experimental condition and pooled from three separate experiments; A, C, one-way ANOVA with post hoc Tukey's, **< 0.01; ***< 0.001). Scale bar, 20 μm.

The heightened level of trkA activation likely has ramifications on both short- and long-term sensitization processes as it regulates the activity and expression of a wide variety of receptors, ion channels, and signaling molecules (Mantyh et al., 2011). In the short term, it modulates the activity of receptors and ion channels. For example, NGF decreases the threshold of activation of the nociceptive transient receptor potential vanilloid 1 (TRPV1) receptors (Chuang et al., 2001), mediates TRPV1 trafficking to the membrane (Stein et al., 2006), increases purinergic receptor P2X3-mediated currents and Ca²⁺ transients (D'Arco et al., 2007) and for sympathetic neurons, rapidly modulates the activity of at least four voltage-gated currents (Luther and Birren, 2009). In the long term, increased NGF can lead to increased transcription of many nociception-associated genes such as its receptors trkA and p75 (Verge et al., 1989, 1992); the neuropeptides Substance P, calcitonin gene-related peptide (Lindsay and Harmar, 1989; Verge et al., 1995), and PACAP; Jongsma Wallin et al., 2001, 2003); sodium channels (Dib-Hajj et al., 1998; Fjell et al., 1999; Kerr et al., 2001); and P2X3 (Ramer et al., 2001; Simonetti et al., 2006). It is also interesting to note that activation of trkA was linked in our study to a parallel activation of p38MAPK, which has been shown to be linked to NGF-mediated increases in ASIC3 expression during inflammation (Mamet et al., 2003).

Related Reagents:
All Trk Antibodies
Neurotrophin Antibodies
Neurotrophin Proteins
Primary Neurons and Astrocytes-Primary human, rat and mouse neurons and astrocytes by Category

More on the Neurobiology of Itch

I have an earlier posting on neuro-transmission of itch or pruritis. This focused on the role of Toll-like Receptor 3 (TLR 3). This posting looks at the role of Gastrin-Releasing Protein (GRP) and the Gastrin-Releasing Protein Receptor (CRPR) in chronic itch. The findings are important because this condition affects millions worldwide and results in a costly erosion of quality of life.

Here chronic itch was studied in Macque Monkeys over a period of 4 years. The expression patterns of GRP, GRPR and PGP9.5 were accessed by immunohistochemistry: Leigh A Nattkemper, Zhong-Qiu Zhao, Anna J Nichols, Alexandru D P Papoiu, Carol A Shively, Zhou-Feng Chen and Gil Yosipovitch. Over-Expression of the Gastrin-Releasing Peptide in Cutaneous Nerve Fibers and its Receptor in Spinal Cord in Primates with Chronic Itch. Journal of Investigative Dermatology accepted article preview 4 April 2013; doi: 10.1038/jid.2013.166.

Images: Double labeling of PGP9.5 and GRP in skin of primates representing mild, moderate and severe itch. Primates with higher scratching severity showed in increase in the co-localization of PGP9.5 and CRP at the dermal-epidermal junctions (arrows).

In addition to increased PGP9.5/GRP expression in the skin, similar results were shown for expression of GRP/GRPR in DRGs. This makes GRP and its receptor candidate drug targets for chronic itch or pruritis in humans.

Neuron-Glial Cultures-Setting a Higher Bar!

Improved Methods for Long Term, High Denisty Cultures

 

Dr. Randen Patterson and his team at UC Davis have developed new culturing techniques using our e18 Rat Primary Hippocampal Neurons. They have developed a protocol that allows for culturing of E18 hippocampal neurons at high densities for more than 120 days. These cultured hippocampal neurons are (i) well differentiated with high numbers of synapses, (ii) anchored securely to their substrate, (iii) have high levels of functional connectivity, and (iv) form dense multi-layered cellular networks. We propose that our culture methodology is likely to be effective for multiple neuronal subtypes–particularly those that can be grown in Neurobasal/B27 media. This methodology presents new avenues for long-term functional studies in neurons. This is good news indeed: Todd GK, Boosalis CA, Burzycki AA, Steinman MQ, Hester LD, et al. (2013) Towards Neuronal Organoids: A Method for Long-Term Culturing of High-Density Hippocampal Neurons. PLoS ONE 8(4): e58996. doi:10.1371/journal.pone.0058996.

 

Protocol Highlights: 
Substrate Preparation
1. On the day of plating, prepare 25 mm coverslips by removing them from 70% ethanol storage solution and propping them up at an angle in each well of a 6-well culture plate to allow drying. [No more than 5 plates (30 coverslips) should be dried simultaneously for 15–25 minutes in culture hood to avoid over-drying.]
2. Once dry, shake slips down flat into their respective wells and coat with 1 mL 0.1% poly-D-lysine, taking care to form a liquid meniscus on each slip. Carefully transfer coverslips into incubator, taking care to preserve meniscus.
3. Incubate for 1 hr at 37°C. [Keeping poly-D-lysine meniscus on top of coverslip is important; this serves to avoid poly-D-lysine coating under coverslip surface that may lead to problematic flotation of coverslip.] After incubation, remove poly-D-lysine and rinse each coverslip three times with 2 mL sterile deionized water. Take care to ensure coverslips do not completely dry at any point during the rinse. After the third and final rinse, leave coverslips in 2 mL sterile deionized water for at least 1 hr. Remove water just before plating, again, make sure to avoid over-drying. [This critical step requires attention. Take care to aspirate off all sterile water remaining from the final rinse, but also use caution as not to over-dry the coverslips. Ultimately, the coverslip must be mostly dry as to allow for the meniscus formation during plating (Fig. 1, Step 12), whereas over-drying can result in the neurons peeling off the glass coverslips days to weeks after plating.]

Fig 1: Neuron-Glial Culturing Steps
Preparation of Isolated Neurons (Numbers in Fig. 1 correspond to numbers below).
1. Store tissue at 4°C until ready to use. If dissecting your own cultures, upon isolation of the tissue, store in an appropriate storage media.
2. When ready to plate, make 2 mL of enzymatic solution without B27. In our case, we used Hibernate E-Ca, containing 4 mg (2 mg/mL) of papain. If making your own solution, use a commercially available papain dissociation kit. Make sure to sterile filter solution with 0.2 micron filter after adding papain if source of enzyme is not sterile.
3. Remove the storage media from the dissected tissue and transfer into sterile 15 mL screw-cap tube; be careful not to disturb or remove tissue from original tube. Save the storage media, do not discard.
4. Add 2 mL of media made in Step 2 to tissue (in our case, Hibernate E-Ca containing 2 mg/mL of papain). Incubate for 35 min at 37°C. [Be sure to add Hibernate E-Ca containing papain slowly as to avoid disturbing tissue.]
5. Remove enzymatic solution from tissue, again, take care not to disturb or remove tissue. Add back 1 mL of storage media saved in 15 mL tube.
6. Using a 1 mL pipettor with a sterile plastic pipette tip (tissue can adhere to glass pipettes), aspirate the tissue with the medium into the pipette and immediately dispense contents back into same container. Take care not to create bubbles. [This is another critical step that requires attention. Take care to make sure pipette tip remains in a stable position (as shown in Fig. 1, Step 6). Maintain slow, steady speed when both drawing in and re-dispensing media containing tissue.]
7. Repeat this trituration step 10–12 times or until most all the tissue is dissociated and the cells are dispersed. [Under close examination cell dispersion is highly visible. Stop pipetting immediately upon cell dispersion.]
8. Slowly transfer contents of the tissue tube into a new sterile 15 mL screw-cap tube.
9. Use the remainder of storage medium saved in Step 3 and rinse the interior of the tissue tube before adding it to the sterile 15 mL screw cap tube containing dispersed cells from step 7. [This step helps ensure minimal wastage, as any remaining cells should be saved with this extra rinse.]
10. Spin dispersed cells at 1,100 rpm (200Xg) for 1 min.
11. Discard the supernatant while being careful not to remove any of the cells from cell pellet.
12. Flick tube a few times to loosen the cell pellet. Re-suspend pellet in 2.4 mL of pre-warmed B27/Neurobasal/0.5 mM glutamine medium. Re-suspend by gently pipetting up and down. For E18 Hippocampus, medium includes 25 µM glutamate.
13. Plate cells within a meniscus (approx. 10 mm diameter) at a minimum of 40 µL per 25 mm coverslip. Take care not to disturb meniscus. [Periodically pipette up and down throughout plating process (no more than once every plate per 6 coverslips) to help maintain equal cell density. Again, plating with meniscus formation is critical.]
14. Incubate plated cells at 37°C with 5% CO2 and/or 9% or 20% oxygen for 1 hr.
15. Add 1.5 mL per well of pre-warmed 1:1 ACM/NbActiv4. [Slow and steady media addition rate and proper pipette position are necessary for successful plating density consistency. Position pipette tip at 45° angle along middle of 6-well interior sidewalls, dispense 1.5 mL as slowly and steadily as possible (see Steps 1–14).]
16. Incubate cells at 37°C with 5% CO2 and/or 9% or 20% oxygen.
17. Add Cytosine β-D-arabinofuranoside (Ara-C) to a final concentration of 5 µM, 5–6 days after plating to curb glial proliferation. [Remove 1/3 of media from each well and replace with equal volume containing final concentration of Ara-C]
18. After 4 days or longer, neurons are well differentiated. If further culture is desired, change 1/3 of medium with fresh, pre-warmed 1:1 ACM/NbActiv4 every 7–8 days.

Images: 40X Confocal Images of 30 DIV Hippocampal Cultures. Immunofluorescence detection of MAP-2 (green) and GFAP (red) in 30 DIV (A–C) cultured E18 hippocampal cells using a 40X objective. These images (A and B) clearly depict the intimate physical contact between glia processes and dendritic arbors. Under closer examination (CI and CII), it is clear that the dendrites have grown both bellow (blue arrows) and above (white arrows) glial processes, forming a highly interconnected three-dimensional network by 30 DIV. doi:10.1371/journal.pone.0058996.g004.
All Primary Neuron Assay Customer Publications

 

Related Content:

• Primary Neurons and Astrocytes-Primary human, rat and mouse neurons and glia/astrocytes.
• Neuronal-Glial Markers-Astrocytes, Glia, Microglia, Olidogodendrocytes, Progenitors and Schwann Cell Markers
• Cells, Cell Culturing Systems and Cell Based Assay Reagents-Cells, Petaka Cell Culturing Systems, Biolumonics Kits, Media, 3-D Scaffolds and Related Reagents Designed to Optimize Cell Based Assays

If you have any questions on optimizing your cell cultures, do not hesitate to contact me @ pshuster@neuromics.com or 612-801-1007

Small Molecules-Peptides for Neuroscience Research

Agonists, Antagonists, Inhibitors and Ligands for Studying Neuromodulation

Our friends at R and D Systems/Tocris Bioscience have made available to us select Small Molecules/Peptides. Our focus will be on providing agonists, antagonists, inhibitors and ligands that complement our Neuroscience and Pain Research products and expertise.

2-APB=TRP Channel Modulator

We will be adding about 10 new molecules/peptides per month. Here's a sampling our our most recent additions:

Name	Type	Bioactivity
(±)-trans-ACPD	Agonist	Potent NMDA agonist. Also group II mGluR agonist
(S)-(-)-5-Fluorowillardiine	Agonist	Very potent AMPA agonist
(S)-4-Carboxyphenylglycine	Antagonist	Competitive group I mGluR antagonist/weak group II agonist
2-APB	Modulator	TRP channel modulator. Also IP3 receptor antagonist
2-Methylthioadenosine triphosphate tetrasodium salt	Agonist	P2 purinergic agonist
AM 404	Modulator	Vanilloid receptor agonist. Also anandamide transport inhibitor
BRL 52537 hydrochloride	Ligand	Potent and selective κ opioid receptor agonist
CNQX	Antagonist	Potent AMPA/kainate antagonist
Clocinnamox mesylate	Antagonist	Irreversible μ-opioid receptor antagonist
Endomorphin-1	Agonist	Potent and selective μ opioid receptor agonist
Endomorphin-2	Agonist	Potent and selective μ opioid receptor agonist
FIT	Agonist	Irreversible δ opioid receptor agonist
GBR 13069 dihydrochloride	Agonist	Potent dopamine uptake inhibitor
L-NIO dihydrochloride	Inhibitor	Potent eNOS inhibitor
L-Quisqualic acid	Agonist	Very potent group I mGluR agonist
N-Benzylnaltrindole hydrochloride	Agonist	Opioid receptor selective non-peptide antagonist
Nociceptin	Inhibitor	Endogenous NOP agonist
O-Phospho-L-serine	Antagonist	Group III mGluR agonist; enhances neuronal differentiation
Ro 51	Antagonist	Potent P2X3, P2X2/3 antagonist
cis-ACPD	Agonist	Potent NMDA agonist. Also group II mGluR agonist

We will be posting new additions and related data and publications