Monday, December 31, 2012

Live Cell Imaging

My friends at Essen Bioscience are taking live cell imaging to new heights. I previously posted their neurite outgrowth solutions (hippcampal neurons).
In this posting, I would like share highlights from the December 2012 issue of "Genetic and Biotechnology News": Essen BioScience’s IncuCyte ZOOM™(live cell imaging in your incubator) and CellPlayer™ reagents enable you to overcome the limitations of static cell based assays. The solutions enable the acquisition, analysis, and quantification of images from living cells that remain unperturbed by the detection method, allowing for repeated measures of cell biology over long periods of time, from days to weeks. This is true Live Content Imaging.

Example: Real-Time Cell Counting in Mixed Cultures.
Cell-based models composed of more than one cell type in the same culture are increasingly recognized as more biologically relevant than monocultures. For example, a recent cancer study illustrated that some stromal cell types confer resistance to tumor cells in coculture, proposing this as a possible mechanism
of tumor resistance in the clinic.
Integrated image processing algorithms provided an independent nuclear count of both cell types continuously in time. The combined attributes of this approach can be used to better elucidate the mechanism and timing of drug responses on cell proliferation in biologically relevant, mixed culture systems.

Capabilities extend to a wide range of other phenotypic assays, including cell death, angiogenesis, neurite dynamics, and cell migration and invasion.

These solutions are have the ability to save time and drive costs out of the drug discovery process. I will keep you posted.

Friday, December 28, 2012

Ischemic Conditioning Prevents Retinopathy

What is Ischemic Conditioning?
I must confess that I had little knowledge of Ischemic Conditioning and its therapeutic potential before accessing this publication by my friend +Laura A. Pasquini and her team at University of Buenos Aires (users of Neuromics' Neuronal-Glial Markers and Neurotrophins Antibodies.

In the conditioning or pre-conditioning process, blood supply to an organ or a tissue is impaired for a short time (usually less than five minutes) then restored so that blood flow is resumed, and the process repeated two or more times, the cells downstream of the tissue or organ are robustly protected from a final ischemic insult when the blood supply is cut off entirely and permanently.

Here the authors used pressure pulses to induce retinal ischemia. Their results suggest that early vision loss in diabetes could be abated by ischemic conditioning which preserved axonal function and structure: Diego C. Fernandez, Laura A. Pasquini, Damián Dorfman, Hernán J. Aldana Marcos, Ruth E. Rosenstein. Ischemic Conditioning Protects from Axoglial Alterations of the Optic Pathway Induced by Experimental Diabetes in Rats. Research Article | published 20 Dec 2012 | PLOS ONE.

They used our PDGFR-α and an O1 marker to compare conditioned, diabetic with unconditioned, diabetic and controls rats to determine protection on ONs of the eye.

Immature OL (O1+ cells) and OL precursor (PDGFR-α+ cells) were evaluated by immunostaining in transverse ON sections. In the diabetic ON from eyes that received a sham treatment, a significant increase in O1(+) and PDGFR-α (+) area was observed, with the presence of disorganized and hypertrophic cells. In the right panel, the area occupied by glial cells (measured as total optical density (OD)) is shown. Ischemic conditioning significantly prevented these alterations and a clear decrease in O1- and PDGFR-α-immunoreactivity, with cells aligned parallel to axon bundles were found. Data are mean ± SEM (n = 6 nerves/group).

Results suggest that early vision loss in diabetes could be abated by ischemic conditioning which preserved distal axonal function and structure before the neuronal soma loss. Moreover, the present results add new potentialities to the therapeutic effects of ischemic tolerance, which is axon protection. Thus, ischemic tolerance could have promise for application in other neurodegenerative axonal diseases. I will keep you updated on progress.

Sunday, December 16, 2012

P2X Receptor Markers-Pubs Update

2012 has been a record year for publications referencing use of our Purinergic Receptor Antibodies. These publications demonstrate use of these markers in a variety of assays and applications. Examples range from analysis of P2X2 expression in human bladder epithelial cells to showing P2X3 expressing nerve fiber boutons in rat horizontal spinal cord sections by immunofluorescence and much more.

Here's a summary of publications: F.C. Pradoa, D. Araldia, A.S. Vieiraa, M.C.G. Oliveira-Fusarob, C.H. Tambelia, C.A. Parada. Neuronal P2X3 receptor activation is essential to the hyperalgesia induced by prostaglandins and sympathomimetic amines released during inflammation. non-fat dry milk at room temperature, followed by incubation with P2X3 or PKCɛ rabbit polyclonal antibody (1:500; Neuromics) overnight at 4 °C, rinsed six times with TBST, and then incubated for 40 min in goat anti-rabbit IgG peroxidase conjugate...

Min Liu, PhD, MD, Yun-fei Xu, PhD, MD, Yuan Feng, PhD, MD, Fengqiang Yang, MD, Jun Luo, MD, Wei Zhai, PhD, MD, Jian-ping Che, MD, Guang-chun Wang, MD, Jun-hua Zheng, PhD. Epigallocatechin gallate attenuates interstitial cystitis in human bladder urothelium cells by modulating purinergic receptors. Journal of Surgical Research.
...P2X2 (Neuromics, Northfield, MN)...

Abeer W Saeed, Alfredo Ribeiro-da-Silva. Non-peptidergic primary afferents are presynaptic to neurokinin-1 receptor immunoreactive lamina I projection neurons in rat spinal cord. Molecular Pain 2012, 8:64 doi:10.1186/1744-8069-8-64Anna M.W. Taylora, Maria Osikowicza, Alfredo Ribeiro-da-Silva. Consequences of the ablation of nonpeptidergic afferents in an animal model of trigeminal neuropathic pain. PAIN. Volume 153, Issue 6, June 2012, Pages 1311–1319.
Confocal images at high power obtained from horizontal spinal cord sections. In a confocal optical section from lamina I adjacent to the white matter (A), note the relatively abundant P2X3-IR fibers with varicosities (boutons). CGRP-IR fibers and boutons were considerably more abundant in this lamina. In a confocal optical section from inner lamina II (B), note the very high density of P2X3-IR fibers and varicosities, higher than that of CGRP-IR fibers in lamina I. Note that most varicosities display either P2X3 or CGRP immunoreactivity, although some co-localization is observed (yellow). Scale bar (A, B) = 20 μm.
T. Cho, V. V. Chaban. Interaction Between P2X3 and Oestrogen Receptor (ER)α/ERβ in ATP-Mediated Calcium Signalling In Mice Sensory Neurones. Journal of Neuroendocrinology Volume 24, Issue 5, pages 789–797, May 2012...with polyclonal rabbit antibody against P2X3 receptor (dilution 1 : 1000; Neuromics)...

Anna M.W. Taylora, Maria Osikowicza, Alfredo Ribeiro-da-Silva. Consequences of the ablation of nonpeptidergic afferents in an animal model of trigeminal neuropathic pain. PAIN. Volume 153, Issue 6, June 2012, Pages 1311–1319. were then incubated for 48 hours at 4°C with a guinea pig polyclonal anti-P2X3 antibody (1:25,000; Neuromics, Edina, MN, USA), diluted in PBS-T. Following primary antibody incubation, sections were treated with a biotin-conjugated...

Ji Z-G , Ito S , Honjoh T , Ohta H , Ishizuka T , et al. 2012 Light-evoked Somatosensory Perception of Transgenic Rats That Express Channelrhodopsin-2 in Dorsal Root Ganglion Cells. PLoS ONE 7(3): e32699...guinea-pig anti-P2X3 (1:1,000, GP10108, Neuromics, Edina, MN, USA)...
Distribution of ChR2V in the dorsal part of the spinal cord. A–C. Immunohistochemical localization of ChR2V with the cell-type specific markers, NF200 (A), CGRP (B) or P2X3 (C). Scale bars indicate 40 µm. doi:10.1371/journal.pone.0032699.g003.

I will continue to post new publications and data in the coming year. Stay tuned. 

Monday, December 10, 2012

Pain Research and Gene Expression Analysis

There have been multiple publications referenced here on using Neuromics' i-Fect siRNA Delivery Kit to study the effect of silencing genes known to play a role in Pain Signaling. These include: DOR,The β3 subunit of the Na+,K+-ATPase, NTS1, NAV1.8, TRPV1 NOV, β-arrestin, TRPV1, CAV1.2 and ASIC.

I would like to highlight an exciting new study referencing how knocking down Kv9.1 Potassium Channel Subunit in vivo mediates neuropathic pain: Christoforos Tsantoulas, Lan Zhu, Yasin Shaifta, John Grist, Jeremy P. T. Ward, Ramin Raouf, Gregory J. Michael, and Stephen B. McMahon. Sensory Neuron Downregulation of the Kv9.1 Potassium Channel Subunit Mediates Neuropathic Pain following Nerve Injury. The Journal of Neuroscience, 28 November 2012, 32(48): 17502-17513; doi: 10.1523/​JNEUROSCI.3561-12.2012.

Highlights: Here, we report that the potassium channel subunit Kv9.1 is expressed in myelinated sensory neurons, but is absent from small unmyelinated neurons. Kv9.1 expression was strongly and rapidly downregulated following axotomy, with a time course that matches the development of spontaneous activity and pain hypersensitivity in animal models. Interestingly, siRNA-mediated knock-down of Kv9.1 in naive rats led to neuropathic pain behaviors. Diminished Kv9.1 function also augmented myelinated sensory neuron excitability, manifested as spontaneous firing, hyper-responsiveness to stimulation, and persistent after-discharge. Intracellular recordings from ex vivo dorsal root ganglion preparations revealed that Kv9.1 knock-down was linked to lowered firing thresholds and increased firing rates under physiologically relevant conditions of extracellular potassium accumulation during prolonged activity. Similar neurophysiological changes were detected in animals subjected to traumatic nerve injury and provide an explanation for neuropathic pain symptoms, including poorly understood conditions such as hyperpathia and paresthesias. In summary, our results demonstrate that Kv9.1 dysfunction leads to spontaneous and evoked neuronal hyperexcitability in myelinated fibers, coupled with development of neuropathic pain behaviors.

n vivo RNA interference: Anesthetized rats were subjected to a thoracic laminectomy and a silastic tube was inserted subdurally to lie just rostral to L3 DRG and externalized to deliver bolus injections (one injection per day for 4 consecutive days). Animals were allowed to recover for 5 d before treatment commenced. On the day of injection, siRNA was mixed with i-Fect (Neuromics) to a final concentration of 0.2 μg μl−1, according to published protocols (Luo et al., 2005). For each treatment, 10–20 μl of Kv9.1 siRNA or scrambled control mixture was injected, followed by a 10 μl saline flush. Twenty-four hours after the fourth injection animals were killed and L5 DRGs fresh dissected for qRT-PCR analysis. A separate set of animals were PFA perfused and DRGs retrieved for IHC. Passenger strand sequences for Kv9.1 and scrambled control siRNAs were cuuggaaucuguaggauca and gaggcctaatcgatatgtt, respectively (Dharmacon; “in vivo processing” option).
Intrathecal Kv9.1 siRNA treatment induces pain behaviors in naive rats. A, qRT-PCR quantification of Kv9.1 mRNA in rat PASMC cultures transfected with one of three Kv9.1 siRNA sequences or control siRNA (control, n = 6; siRNA, n = 3 per group; *p < 0.05 vs control, one-way ANOVA with Tukey's). B, qRT-PCR showing Kv9.1 in vivo knock-down in L5 DRG, 4 d after intrathecal delivery of siRNA #1 compared with vehicle or matched scrambled control (vehicle, n = 4; scrambled, n = 5; Kv9.1, n = 7; *p < 0.05, t test). C, IHC for Kv9.1 in scrambled- and siRNA-treated DRG to determine protein knockdown. Graphs illustrate quantification of number of positive myelinated neurons and mean Kv9.1 signal intensity (scrambled, n = 4; siRNA, n = 6; **p < 0.01, ***p < 0.001, t test). D, Kv9.1 siRNA infusion inflicts a reduction in mechanical pain withdrawal thresholds (Kv9.1, n = 7; control, n = 6; *p < 0.05, **p < 0.01, ***p < 0.001 vs scrambled control or baseline, two-way repeated measurements ANOVA with Tukey's). E, There was no change in heat pain thresholds after siRNA treatment. Vertical arrows on x-axis denote siRNA injections. All data represent mean ± SEM.
Kv9.1 knock-down triggers ectopic activity and a form of peripheral wind-up in response to stimulation. A, Schematic illustrating the positions of stimulating and recording electrodes. B, Example recordings from centrally disconnected L4/L5 strands demonstrating SA in Kv9.1 siRNA-treated or nerve-injured rats, but not in control (scrambled siRNA) animals. C, Frequency-dependent SEA (denoted by double arrowheads) in Kv9.1 siRNA-treated (middle) and injured (right), but not control (left) animals. This activity is not locked in time and can be seen in between stimulation events (vertical arrows on top of 5 Hz stimulation traces, only first 5 shown). Also note the prolonged after-discharge (AD) observed in siRNA-treated and injured animals. D, Percentage of units showing SA and SEA in control (n = 269), Kv9.1 siRNA-treated (n = 369) and injured (n = 176) animals (*p < 0.05, **p < 0.01, ***p < 0.001 vs control, χ2 test). E, Firing rate of SEA units at different stimulation frequencies (mean ± SEM; control, n = 4; siRNA, n = 22; injured, n = 17; *p < 0.05 vs control, two-way ANOVA with Tukey's). F, Quantification of AD rate per SEA unit (mean ± SEM; *p < 0.05 vs control, Mann–Whitney test).

Results propose that Kv9.1 downregulation after nerve injury may be the molecular switch controlling myelinated sensory neuron hyperexcitability. Intriguingly, a recent wide-genome association screen in humans identified a Kv9.1 polymorphism associated with susceptibility to develop chronic neuropathic pain after back surgery or leg amputation (Costigan et al., 2010), suggesting that the mechanisms described in our studies will be of direct clinical relevance to human pain. Future efforts to elucidate the precise pathways involved, combined with approaches aiming to compensate loss of Kv9.1 function, may create novel therapeutic opportunities for neuropathic pain management.

Wednesday, November 21, 2012

Oncolytic viruses=Cancer Killers

Oncolytic viruses, including vaccinia virus (VACV), are a promising alternative to classical mono-cancer treatment methods such as surgery, chemo- or radiotherapy. However, combined therapeutic modalities may be more effective than mono-therapies. In this study, researchers enhanced the effectiveness of oncolytic virotherapy by matrix metalloproteinase (MMP-9)-mediated degradation of proteins of the tumoral extracellular matrix (ECM), leading to increased viral distribution within the tumors.

Protein expression  levels were determined using Neuromics' Goat Polyclonal MMP-9 antibody: Simon Schäfer, Stephanie Weibel, Ulrike Donat, Qian Zhang, Richard J Aguilar, Nanhai G Chen and Aladar A Szalay. Vaccinia virus-mediated intra-tumoral expression of matrix metalloproteinase 9 enhances oncolysis of PC-3 xenograft tumors. BMC Cancer 2012, 12:366 doi:10.1186/1471-2407-12-366.

Images and Figures: MMP-9 expression in PC-3 tumor sections and collagen IV quantification. Intratumoral expression of MMP-9 (red) was visualized using MMP-9 labeled PC-3 tumor sections. Nuclei (white) were stained with Hoechst 33258 dye. GFP (green) is a VACV reporter gene. Tumors were obtained at 7 days p.i. from PC-3 tumor-bearing mice injected with PBS, GLV-1h68 or GLV-1h255. All images are representative examples. (B) Quantification of MMP-9 expression was done by microscopic analysis. Mean fluorescence intensities were measured with ImageJ. (C) Quantification of collagen IV 7 days p.i. in GLV-1h68 or GLV-1h255 infected areas of PC-3 tumor sections. Images were taken at a 100× magnification (Leica MZ16 FA) and converted from RGB to grayscale using Photoshop. For image analysis ImageJ was used, the threshold value was 8/255.

Figure and Images: Expression of functional MMP-9 by GLV-1h255-infected tumor cells. (A) Expression cassettes of GLV-1h68 and GLV-1h255. In GLV-1h255 the insert in the Tk locus was replaced by the human mmp-9 gene under control of the PSE promoter. PSEL, synthetic early/late promoter; PSE, synthetic early promoter; P7.5, VACV p7.5 K early/late promoter; P11, VACV p11 late promoter; Tk, thymidine kinase locus, Ha, hemagglutinin locus. (B) Expression of virus-encoded MMP-9 (92 kDa) in GLV-1h255 infected PC-3 cells and supernatants in vitro, β-actin (42 kDa) was used as a loading control. (C) Activity of the MMP-9 protein was tested by gelatin zymography. Lysates and supernatants of infected A549 cells were isolated and separated by non-reducing SDS-PAGE. In zymography, cleavage of the substrate by MMP-9 resulted in a clear band.

This study revealed that the degradation of collagen IV (ECM) by VACV-encoded MMP-9 may represent a new option to significantly enhance the oncolytic effect of rVACV in PC-3 xenografts. We confirmed that the degradation of collagen IV facilitated viral infection of the tumor tissue, represented by significantly higher viral tumor titers and an accelerated tumor regression. Furthermore, both oncolytic viruses, parental GLV-1h68 and mmp-9-encoding GLV-1h255, significantly reduced the size of lumbar and renal lymph node metastases, indicating that MMP-9 enhances both virotherapy of the primary tumor and sustains the rVACV-metastasis reducing effect.

I will keep you posted on further studies.

Saturday, November 10, 2012

More On Petaka Mini Bioreactors

Getting Culture Conditions Right Every Time

I would like to provide yet more information on the capabilties of our new and innovative Petaka Mini Bioreactors. This posting focuses how oxygen concentrations are tightly controlled in a way that is consistent with the cultured cells natural environment.

The length and cross-section of the respiratory duct is purposely engineered to partially restrict the diffusion of oxygen from the high levels of ambient air to create lower, physiologic levels of dissolved oxygen in the reaction chamber. In accordance to Fick’s Law, as oxygen is consumed inside the culture chamber, decreasing the partial pressure of oxygen in the media, oxygen diffuses in from the outside atmospheric (higher) partial pressure, through the respiratory duct, to the lower partial pressure inside. Diffusion is proportional to the concentration gradient, as regulated by the engineered design of the respiratory duct, and occurs entirely spontaneously and without any manual intervention whatsoever.

Image: Petaka®G3 Ducted Respiratory Chamber (DRC). (1) cell culture chamber;(2) injection port; (3) respiratory duct; (4) 0.2μ filter; (5) water vapor condensers and capillary breakers; (6) unique barcode. The DRC is shown upright in a silicone stand (7).

At the same time, the respiratory duct partially retains carbon dioxide from cellular metabolism to maintain a physiologically normal mild acidosis to balance pH. All gas exchange with the outside environment occurs via a 0.2 micron filtered vent, preserving the internal sterility of the device but allowing exchange of gas diffusion and flow to prevent pressurization issues when filling and emptying the bioreactors.
Therefore, cell culture in DRC’s/Petaka G3 does not require supplemental oxygen-nitrogen balancing, CO2 and humidity sources, eliminating the entire panoply of gas tanks, regulators, sensors, microprocessors and water pans. This creates a double benefit: not only are cells cultured in more normal physiologic conditions, but the mechanics, logistics, risks and costs of cell culture are greatly simplified and reduced.

The Results are Stunning!

Here are result using Petaka® G3 LOT for culturing our UCB Derived hMSCs and Mouse MSCs.
Images: (A) Immuno-fluorescence microscopy of a mouse MSC in differentiation progression. Culture under 20 mmHg of O2 partial pressure. Red fluorescence positive staining of GFAP. hMSC 3 hours after seeding in Petaka G3 with low serum media and without matrix (B) and 96 h later (C). Photos: Jim Musick. Vitro-Biopharma. September, 2012.

Next up using Petaka DRCs for GMP. Stay tuned.

Sunday, November 04, 2012

True Physiologic Conditions in Cell Culturing

The Petaka Advantage!

A cornerstone of Neuromics' strategy is to enable better science by finding ways to help our customers and collaborators to both improve and lower the overall costs of their cell based assays.

We embrace new technologies if they prove capable of providing cell culture environment that more closely mimic in vivo environments. This enables basic and drug discovery researchers learn more about the true potential of targets. More informed decisions early in the process reduce downstream costs.

This is why the use of engineered mini bioreactors with Ducted Respiratory Chambers (DRCs) like the Petaka® G3 LOT needs to be considered. (detailed information @ Ducted Respiratory Chamber Bioreactors.© 2012 Genetic Engineering & Biotechnology News All Rights Reserved). Instead of attempting to impose “normal” gas conditions on the cells through active incubator controls, this system passively allows cells to maintain their own oxygen, carbon dioxide, and humidity levels.
Image: Petaka's DRC design. The cell culture chamber is isolated on the injection side from the atmosphere by a self-sealing silicone injection port that allows the closed introduction of media and cells, including most eukaryotic cells types, small early-stage embryos, tissue fragments, and even needle biopsies.

Because the cells control their own gas environment, there is little effect from outside gas conditions, and the DRC is effective in atmospheres from 500 meters below sea level up to elevations of 4,000 meters, and in relative humidity levels between 10% and 100%.
Figure: Self-regulating gas management in the DRC. Cells consume O2, with restricted O2 ingress, causing a first proliferative cell phase to evolve into a second differentiating cell phase for protein production and gene expression.

The DRC changes the paradigm of cell culture, replacing nearly a century of active attempts to humanly intervene to manipulate gas exchange with a self-regulating design driven by natural laws of diffusion to create dependable, and truly physiologic, gas environments.

Monday, October 29, 2012

OPC Markers!

Effective Oligodendrocyte, Oligodendroglial Oligodendrocyte Lineage Markers are important for determining the differentiate state of Oligodendrocyte Precursor Cells. This is important for the study of de and re-myelination of neurons and the discovery of potential therapeutic targets for diseases like MS and ALS.

Here researchers use our Olig2 antibody to study the differentiation state of Fetal Human Oligodendrocyte Progenitor Cells: Crystal R. McClain, Fraser J. Sim and Steven A. Goldman. Pleiotrophin Suppression of Receptor Protein Tyrosine Phosphatase-β/ζ Maintains the Self-Renewal Competence of Fetal Human Oligodendrocyte Progenitor Cells. The Journal of Neuroscience, 24 October 2012, 32(43): 15066-15075; doi: 10.1523/​JNEUROSCI.1320-12.2012.
Abstract: Oligodendrocyte progenitor cells (OPCs) persist in human white matter, yet the mechanisms by which they are maintained in an undifferentiated state are unknown. Human OPCs differentially express protein tyrosine phosphatase receptor β/ζ (PTPRZ1) and its inhibitory ligand, pleiotrophin, suggesting the maintenance of an autocrine loop by which PTPRZ1 activity is tonically suppressed. PTPRZ1 constitutively promotes the tyrosine dephosphorylation of β-catenin and, thus, β-catenin participation in T cell factor (TCF)-mediated transcription. Using CD140a/PDGFRα-based fluorescence-activated cell sorting to isolate fetal OPCs from the fetal brain at gestational ages 16–22 weeks, we asked whether pleiotrophin modulated the expansion of OPCs and, if so, whether this was effected through the serial engagement of PTPRZ1 and β-catenin-dependent signals, such as TCF-mediated transcription. Lentiviral shRNAi knockdown of PTPRZ1 induced TCF-mediated transcription and substantially augmented GSK3β inhibition-induced TCF-reporter luciferase expression, suggesting dual regulation of β-catenin and the importance of PTPRZ1 as a tonic brake upon TCF-dependent transcription. Pharmacological inhibition of GSK3β triggered substrate detachment and initiated sphere formation, yet had no effect on either proliferation or net cell number. In contrast, pleiotrophin strongly potentiated the proliferation of CD140a+-sorted OPCs, as did PTPRZ1 knockdown, which significantly increased the total number of population doublings exhibited by OPCs before mitotic senescence. These observations suggest that pleiotrophin inhibition of PTPRZ1 contributes to the homeostatic self-renewal of OPCs and that this process is mediated by the tonic activation of β-catenin/TCF-dependent transcription.

Images: To verify that GSK3β inhibition was effecting TCF activation through altering localization of β-catenin, the Wnt signaling intermediate, β-catenin, was localized by confocal imaging in OPCs, validated as such by their coexpression of Olig2.

Marker Options:
NameCatalog #TypeSpeciesApplicationsSizePrice
CNPaseCH23013Chicken IgYH; MICC; IHC100 ul$89
Caspr2SP15104Sheep IgGH; MIHC; WB; E100 ug$365
HSP105MO20028Mouse IgGH; M; RIHC; WB100 ul$155
MAG/Siglec 4aGT15152Goat IgGRIHC; WB; E100 ug$365
MOGGT15141Goat IgGHIHC; WB; E100 ug$365
Mash1GT15216Goat IgGMIHC; WB; E100 ug$365
Mash1MO15048Rat IgGH; MICC; WB; E100 ug$255
NOGO ReceptorGT15154Goat IgGHIHC; WB; E100 ug$365
OMgpGT15200Goat IgGHWB; E100 ug$365
Olig1RA14141Rabbit IgGRIHC100 ul
100 ul @ 1mg/ml
Olig1,2,3MO15059Mouse IgGH; RIHC100 ug$305
Olig2GT15132Goat IgGH; MIHC; WB; E100 ug$365
Olig2RA25081Rabbit IgGH; M; RICC; IHC; WB; IP100 ul$395
Oligodendrocyte Marker O1MO15001Mouse IgMH; M; RIHC; FC50 ug$215
Oligodendrocyte Marker O4MO15002Mouse IgMC; H; M; RIHC50 ug$215
Oligodendrocyte Marker O4-Phycoerythrin LabeledFC15013Mouse IgMHFC100 Tests$305
PDGF R Alpha/CD140AGT15150Goat IgGMIHC; WB; E100 ug$365

Sunday, October 21, 2012

P2X3 Receptor and CGRP Antibodies Immunostaining

Dr. Alfredo Ribeiro-da-Silva, McGill University, is a serial publisher of studies using our pain and inflammation research antibodies.

Here, use of our rabbit anti-CGRP and guinea pig anti-P2X3 is referenced. Please note the high titer of these antibodies (dilution is 1:25,000): Abeer W Saeed, Alfredo Ribeiro-da-Silva. Non-peptidergic primary afferents are presynaptic to neurokinin-1 receptor immunoreactive lamina I projection neurons in rat spinal cord. Molecular Pain 2012, 8:64 doi:10.1186/1744-8069-8-64.

Images: CGRP, IB4 and P2X3 staining in transverse spinal cord sections. A and B show low magnification confocal images of CGRP-IR and IB4 positive (A) or P2X3-IR (B) fibers. C and D represent high magnification confocal images from the middle third of the lateromedial extent of the superficial dorsal horn. In C, note that there is limited co-localization of IB4 and CGRP (in yellow). Arrowheads show axonal varicosities (boutons) from nonpeptidergic fibers in lamina I, which do not co-localize CGRP immunoreactivity. The framed regions in A and B indicate the approximate regions from where C and D, respectively, were obtained (the latter originate from other sections). CGRP (in green); IB4 (in red); P2X3 (in red). Scale bar (A, B) = 200 μm; scale bar (C, D) = 20 μm

Tissue processing: The injection site at the level of the parabrachial nucleus was examined by cutting serial, 100 μm-thick coronal sections of the relevant brain region. The dorsal aspect of the L4-L5 spinal cord segment was cut into serial, 50 μm-thick horizontal sections (n = 10), 50 μm-thick parasagittal sections (n = 4) or 50 μm-thick transverse sections (n = 4). All sections were cut using a freezing sledge microtome (Leica, Richmond Hill, Ontario) and collected as freefloating in phosphate-buffered saline (PBS) with 0.2% Triton-X 100 (PBS + T). To block unspecific staining, all spinal cord sections were incubated, for one hour, in 10% normal donkey serum (NDS) (Jackson, West Grove, PA) in PBS + T at room temperature. Subsequently, the sections were placed in primary antibodies (or conjugated lectin IB4 - see below) for 48 hours at 4 °C. We used a mixture of 2 or 4 primary antibodies (each raised in a different species), or IB4, in PBS + T containing 5% NDS. Next, the sections were washed in PBS + T and then incubated in species-specific secondary antibodies that were raised in donkey and conjugated to either AlexaFluor 488, AlexaFluor 405, Rhodamine RedX or biotin. The sections were incubated in 3 different cocktails: #1) rabbit anti-CGRP at a 1:200 dilution (Sigma, St Louis, MO) and lectin IB4 conjugated to AlexaFluor 568 at a 1:200 dilution (Molecular Probes); #2) rabbit anti-CGRP and guinea pig anti-P2X3 at a 1:25,000 dilution (Neuromics, Edina, MN); #3) goat anti-CTb at a 1:5000 dilution (List Biological), rabbit anti-NK-1r at a 1:10000 dilution (Sigma, St Louis, MO), guinea pig anti-CGRP at a 1:8000 dilution (Peninsula, San Carlos, CA) and lectin IB4 conjugated to AlexaFluor 647 at a 1:200 dilution (Molecular Probes). All the sections were washed with PBS + T and then (for #1) incubated for 2 hours at room temperature with donkey anti-rabbit AlexaFluor 488; (for #2) incubated for 90 minutes in a biotin conjugated donkey anti-guinea pig IgG (Jackson Immunoresearch, West Grove, PA, 1:200). Further signal amplification was achieved by treating the sections with 1 hour incubation in an avidin-biotin (A + B) complex (Vectastain Elite ABC kit, Vector Laboratories) followed by tyramide (Perkin-Elmer, Norwalk, CT, 1:75) for 7 minutes. Sections were then incubated in streptavidin conjugated to AlexaFluor 568 (Molecular Probes, Eugene, OR, 1:200) and donkey anti-rabbit AlexaFluor 488; or (for #3) incubated for 2 hours at room temperature with secondary antibodies: donkey anti-goat Rhodamine Red X, donkey anti-rabbit AlexaFluor 488, and donkey anti-guinea pig AlexaFluor 405. Finally, sections were washed with PBS, mounted on gelatin-subbed slides and coverslipped with an anti-fading mounting medium (Aqua Polymount; Polysciences, Warrington, PA). Slides were stored at −4 °C pending further processing.

I will continue to publish outstanding customer data/images using our natibodies/markers.

Sunday, October 14, 2012

Teminally Differentiated Human Chondrocytes

Save 20% on New Chondrocytes through November 30, 2012

Certain customers tell me they purchase our UCB and eSC Derived Human Mesenchymal Stem Cells to grow and differentiate into Chondrocyte cultures for the study of joint disease.

Our goal is to save time and expense in the development of your cell based assays. We now offer these options:
NameCatalog #TypeSpeciesApplicationsSizePrice
Native Human Chondrocytes-PilotSC00A7-100KPrimary CellsHCell Assays100,000 Cells$245
Native Human Chondrocytes-HCSSC00A7-500KPrimary CellsHCell Assays500,000 Cells$449
Native Human Chondrocytes-HTS PilotSC00A7-4000KPrimary CellsHCell Assays4X1,000,000 Cells$2,399
Native Human Chondrocytes-HTSSC00A7-1000KPrimary CellsHCell Assays1,000,000 Cells$799
Fluorescein Labeled Human Chondrocytes-PilotSC00A8-100KPrimary CellsHCell Assays100,000 Cells$295
Fluorescein Labeled Human Chondrocytes-HCSSC00A8-500KPrimary CellsHCell Assays500,000 Cells$449
Fluorescein Labeled Human Chondrocytes-HTS PilotSC00A7-1000KPrimary CellsHCell Assays1,000,000 Cells$799
Fluorescein Labeled Human Chondrocytes-HTSSC00A7-4000KPrimary CellsHCell Assays4X1,000,000 Cells$2,399
Rhodamine Labeled Human Chondrocytes-PilotSC00A9-100KPrimary CellsHCell Assays100,000 Cells$295
Rhodamine Human Chondrocytes-HCSSC00A9-500KPrimary CellsHCell Assays500,000 Cells$449
Rhodamine Labeled Human Chondrocytes-HTS PilotSC00A9-1000KPrimary CellsHCell Assays1,000,000 Cells$799
Rhodamine Labeled Human Chondrocytes-HTSSC00A9-4000KPrimary CellsHCell Assays4X1,000,000 Cells$2,399
Human Chondrocytes MediaSC00PC3-100Cell Growth MediaH100 ml
500 ml

Images: Chondrocyte cultures.

We plan on continuing to add new potent and pure primary cells to accelerate meaning results from basic disease research and drug discovery.

Wednesday, October 03, 2012

Neuropeptide Ys-Regulate Food Intake & Body Weight

Role of salivary PYY in the modulation of food intake.

This is a study that comprehensively shows the expression of Neuropeptide Ys  (PYY 3–36 ) are present in saliva and showed the expression of its preferred receptor, Y2R, in the basal layer of the progenitor cells of the tongue epithelia and von Ebner's gland. The researchers used our Y2R antibody (dilution 1:3000) to determine level of expression. This receptor appears to be the main player in mediating hunger and body weight: Hurtado MD, Acosta A, Riveros PP, Baum BJ, Ukhanov K, et al. (2012) Distribution of Y-Receptors in Murine Lingual Epithelia. PLoS ONE 7(9): e46358. doi:10.1371/journal.pone.0046358.

Images: Immunolocalization of Y1, Y2, Y4, and Y5 receptors (Rs) in the dorsal epithelium of murine tongue. Images: Mirror section pairs (Panels A and B, C and D, E and F) were hybridized to the respective YR antibody (green), followed by DAPI counterstain (blue), as indicated in the upper left corner of each panel. For better viewing, the confocal images in B, D, and F were reflected horizontally. Representative areas of the epithelium, positive for either YR (dashed rectangles in the left-sided panels), are shown as close-up images on the right next to each respective panel. The irregular columned structures at the epithelial surface are transversely sectioned filiform papillae. G - Y4R-positive neuronal fibers (green) are located in the subepithelial region underlying the basal laminae. H – co-localization of Y4R and NCAM (red) immunoreactivity within mechanoreceptors of Meissner corpuscles (MC). As a morpho-histological reference of the dorsal lingual epithelium structure, an hematoxylin and eosin stained section is shown in panel I. Panel J shows a hypothetical diagram of a lingual dorsal epithelium layer and the differentiation/migration lineage of cell types expressing respective YR subtypes. K5 – cytokeratin-5 [2]. doi:10.1371/journal.pone.0046358.g006.

Note: this study is further validation of the potency of our NPY Y2 antibody.

Conclusion: NPY family peptides and their cognate receptors in the oral cavity may mediate a wide variety of functions, including proliferation, differentiation, motility, taste perception, as well as satiation. All of these multiple functions and their respective molecular mechanisms are subjects of the ongoing investigations.

Understanding these molecular mechanisms could provide the foundation for discovering less intrusive therapies for obesity. I will keep you posted.

Tuesday, September 25, 2012

Cancer Research-Why 3-D Cultures Work

The concept of bringing your cancer cell assay to life with 3-D nanofibers is new. New approaches beg the question, "why change?". Traditional 2-D cultures are the standard and work well enough for many assays.

Change is driven by proof that the new solutions yield better results. I would like to share data showing the potential capabilities and benefits of switching to random and aligned nanofibers.

Increased drug sensitivity

Increased drug sensitivity of human A549 lung cancer cells when grown on random nanofibers or aligned nanofibers when compared to flat tissue culture plastic (TCP). This shows the significant problem of developing drugs using 2-D surfaces and explains why such a large amount of animal testing is required for pre-clinical drug development. Proliferation on 2-D TCPS is artificially high when compared to 3-D culture on nanofibers. Using a high-throughput 3-D nanofiber-based scaffold for in vitro drug screening can more accurately predict the in vivo response of drugs.
The ability to do high-resolution imaging through the nanofiber scaffold (through the bottom of the culture plate) is critical to validate cell/phenotype markers especially in high throughput screening and high content analysis.  Standard microscopes and automated plate readers using light, fluorescence, absorbance, or luminescence are compatible with the nanofiber plates.  Comparison of A549 cells on flat tissue culture polystyrene (A), randomly oriented nanofibers (B), and aligned nanofibers (C).

Cancer Cell Migration-More Like in vivo
Scanning electron microscope images of an ex-vivo human glioblastoma tumor sample cultured on aligned nanofibers showing the tumor dispersion along the nanofibers exactly how they would migrate in vivo along the white matter within the brain and central nervous system.

I will continue to post new developments with our 3-D culturing products. It is our goal to bring your cell based assays to life!

Saturday, September 22, 2012

Neuron-specific class III beta-tubulin (TuJ1) Staining in hNT2.19 Neurons

Our Tuj-1 antibody is widely used and frequently published. It is proving a potent marker for confirming the differentiation of human neural progenitors to neurons.

In this study the authors use the marker for staining hNT2.19 Neurons: Mary J. Eaton, Yerko Berrocal, and Stacey Q. Wolfe. Potential for Cell-Transplant Therapy with Human Neuronal Precursors to Treat Neuropathic Pain in Models of PNS and CNS Injury: Comparison of hNT2.17 and hNT2.19 Cell Lines. Pain Research and Treatment. Volume 2012 (2012), Article ID 356412, 31 pages. doi:10.1155/2012/356412. 
The results show great promise. They show hNT2 or hNT2-derived cell lines, such as hNT2.17 and hNT2.19, have great potential to permanently reverse symptoms of neuropathic pain following PNS and CNS injuries and can offer new hope to treat these intractable conditions to significantly improve human health. This includes neuropathic pain resulting from diabetic neuropathy and Spinal Cord Injury.

Images: Comparison of graft sites of hNT2.17 and hNT2.19 in the QUIS and severe contusive-SCI models, respectively, at 6 weeks after cell transplant. (a) Sagittal section of anti-GABA-immunostained QUIS + hNT2.17 transplant lumbar spinal cord 6 weeks after grafting. Easily detectible hNT2.17 cells stain for GABA (arrows) on the pial membranes. (b) Sagittal section of anti-NuMA-immunostained QUIS + hNT2.17 transplant lumbar spinal cord 6 weeks after grafting. Easily detectible hNT2.17 cells stain for NuMA (arrows) on the pial membranes in adjacent sections. The hNT2.19 were alternately injected into the subarachnoid space two weeks after severe contusive SCI. Cell graft sites were co-localized with 5HT (c) and the human-specific marker TuJ1(d) (neuron-specific class III β-tubulin). There are many surviving hNT2.19 (d) grafted cells visible on the pial surface, which stain for TuJ1 (arrows) at the end of the experiment, 56 days after SCI and about 6 weeks after cell transplant. Adjacent sections with the same grafted hNT2.19 (c) are labeled for 5HT (arrows). Magnification bar = 20 μm.

Protocol: Modified methods for staining spinal cord sections for the human neuron-specific class III beta-tubulin (TuJ1) to identify grafted hNT2.19 neurons after grafting have previously been described [51]. The sections were washed with 0.1 M PBS pH 7.4 and permeabilized with 0.4% Triton-X-100 in 0.1 M PBS, 10% normal goat serum (NGS) for one hour. The sections were then incubated overnight at 4°C in the primary anti-TuJ1 antibody (1 : 100 DPBS), and the permeabilizing solution, followed by a one-hour incubation at room temperature with the secondary antibody solution, biotinylated mouse IgG raised in goat (Vector; 1 : 200), a Peroxidase ABC reporter in 0.1 M PBS (Vector) and “VIP” substrate (Vector). Some sections were stained in the absence of primary antibody and served as the negative controls.

I will continue to post new applications for our stem cell research reagents.

Monday, September 17, 2012

Neuromics' Cortical Neurons & Kinetic NeuroTrack Assays

Providing tools that insure excellent Cell Based Assays is a cornerstone of our business strategy. Lauren McGillicuddy and her team at Essen Bioscience have been using our E18 Primary Rat Cortical Neurons to develop NeuroTrakTM assays enabling kinetic quantification of neurite dynamics (initiation, branching, extension, retraction). NeuroTrack is one of several CellPlayerTM assays that can be run in IncuCyte ZoomTM.

The proof is in the results and these show the both the potency of the cells and the powerful capablities of the IncuCyte ZOOM hardware and software:

Images:  Neurite outgrowth of rat E18 cortical neurons in a 96-well microplate at 24, 48 and 120 hours. top: 20x HD phase image of primary neurons  bottom: Image segmentation of neurites (light blue) and cell body cluster (raspberry).

Images: Neurite outgrowth of rat E18 cortical neurons in a 96-well microplate. Left: 20x HD phase image of primary neurons, 96 hours post plating ; Middle: Image segmentation of neurites (yellow) and cell body cluster (raspberry); Right: Concentration and time dependent inhibition of neurite outgrowth with the protein kinase C inhibitor, Ro-31-8220 (mean ± SD; n=6 per condition).

Check out this cool  assay animation!


I will continue to keep you posted on new assays and related methods using our primary neuron and astroglial cells.

Sunday, September 16, 2012

Focus on Vision Systems Research

New Reagents, Publications and Data

We are intensifying our focus on Vision System Research with the addition of new antibodies. We are offering 50 USD off. They include: PSD-95/SAP90, Rhodopsin (A531), Rhodopsin (B630) and select Calcium (Ca2+) Signaling-Binding antibodies.
Image: Rhodopsin staining of pig retinal sections (green) and counter-stained with NF-M (red) and DNA (blue). Rhodopsin is most abundant in the outer segments of retina (OS), NF-M is abundant in the optic nerve fiber layer (ONFL), but seen in processes and neurons in other regions also. Other layers are pigmented epithelium (PE), outer and inner nuclear layers (ONL, INL), outer and inner plexiform layers (OPL, IPL) and ganglion cell layer (GCL). Inset: Bovine retinal extracts blotted with rhodopsin. Protocols on data-sheet.
Our ability to effectively serve researchers is confirmed by the growing parade of publications. I would like to highlight a recent publication by Dr. Sal Salvatore and his colleagues. It features use of our Shank 1a antibody. They are the first to show Shank 1 expression in the mammalian retina revealing Shank 1 immunoreactivity within both synaptic layers of the retina: Salvatore L. Stella Jr, Alejandro Vila, Albert Y. Hung, Michael E. Rome, Uyenchi Huynh, Morgan Sheng, Hans-Juergen Kreienkamp, Nicholas C. Brecha. Association of Shank 1A Scaffolding Protein with Cone Photoreceptor Terminals in the Mammalian Retina. PLoS ONE 7(9): e43463. doi:10.1371/journal.pone.0043463.

Images: Shank 1A immunoreactivity is in both the inner plexiform layer (IPL) and outer plexiform layer (OPL) of the mouse YFP-16 line retina. A–C: A. Image of a retinal section immunostained for Shank 1A. B. Mouse YFP-16 line vertical retinal section. C. Shank 1A (red) immunolabeling and YFP (yellow). Shank1A expression is restricted to the OPL and IPL. A regular pattern of Shank 1A immunolabeling appears in the OPL, which is indicative of cone photoreceptor terminals. D–E: High magnification zoom of the OPL demonstrates that Shank 1A puncta (red) are distal to the dendrite tips (yellow) of YFP labeled cone bipolar cells, suggesting that Shank 1A is expressed presynaptic to the YFP cone bipolar cell dendrite. G–L: High magnification zoom of the IPL demonstrates that Shank 1A puncta are likely expressed postsynaptically to bipolar cell terminals. G. Shank 1A immunoreactive puncta. H. YFP labeled neurons and processes within the IPL region. I. PKCα labeled rod bipolar cell axons and terminals. J. Combined Shank 1A (red) and PKCα (blue) immunolabeling illustrate that shank 1A puncta are postsynaptic to rod bipolar cell terminals in the IPL. K. Combined Shank 1A (red) immunolabeling and YFP (yellow) in the IPL demonstrate that Shank 1A puncta are postsynaptic to cone bipolar cell terminals in the IPL. L. Combined triple fluorescent image of Shank 1A (red), PKCα (blue), and YFP (yellow) in the IPL. OPL = outer plexiform layer, INL = inner nuclear layer, IPL = inner plexiform layer, and GCL = ganglion cell layer. Scale bars = 10 µm. doi:10.1371/journal.pone.0043463.g001

More Pubs
Diego C. Fernandez, Laura A. Pasquini, Damián Dorfman, Hernán J. Aldana Marcos, Ruth E. Rosenstein. Early Distal Axonopathy of the Visual Pathway in Experimental Diabetes. doi:10.1016/j.ajpath.2011.09.018

...a goat polyclonal anti–platelet-derived growth factor receptor-α (PDGFR-α) antibody (1:100; Neuromics, Edina, MN)
Raoul Torero Ibad, Jinguen Rheey, Sarah Mrejen, Valérie Forster, Serge Picaud, Alain Prochiantz, and Kenneth L. Moya. Otx2 Promotes the Survival of Damaged Adult Retinal Ganglion Cells and Protects against Excitotoxic Loss of Visual Acuity In Vivo. The Journal of Neuroscience, 6 April 2011, 31(14): 5495-5503; doi: 10.1523/​JNEUROSCI.0187-11.2011...For antibody neutralization experiments, anti-Otx2 antibody (Neuromics) was dialyzed against PBS, and then Otx2 (25 ng in 500 mul) was incubated with anti-Otx2 (0.5 mug) in culture medium at 37C...

Hoon Shim, Chih-Ting Wang, Yen-Lin Chen, Viet Q. Chau, Kevin G. Fu, Jianqi Yang, A. Rory McQuiston, Rory A. Fisher, and Ching-Kang Chen. Defective Retinal Depolarizing Bipolar Cells (DBCs) in Regulators of G-protein Signaling (RGS) 7 and 11 Double Null Mice. JBC Papers in Press. Published on February 27, 2012 as Manuscript M112.345751. The latest version is at were sacrificed by CO2 inhalation and the eyeballs were immediately enucleated. After removal of cornea and lens the resulting eyecups were immersionfixed in 4% paraformaldehyde in 1X PBS at room temperature for 15 minutes. This short fixation time ensured good mGluR6 and Gb5 signals at the OPL. After cryoprotection in 30% sucrose in 1X PBS, the eyecups were embedded in TBS (Richard Allan Scientific, Kalamazoo, MI), sectioned at 20μm thickness, and stained...

I will keep you posted on progress.

Wednesday, September 12, 2012

ASIC3 and Osteoarthritis

ASIC3 modulates pain and disease progression

Neuromics' foundation is built on providing reagents for pain researchers. I have posted the twists and turns via key publications and related data. Here's yet another success story with one of our Pain and Inflammation Research Antibodies.

Acid sensing ion channels (ASICs) are sodium-selective ion channels activated by low extracellular pH, and belong to the degenerin/epithelial Na+ channel superfamily. ASIC3  is the most sensitive to such a pH change [2,3], abundantly expressed in dorsal root ganglia (DRG) [4], and strongly correlated with pain. Here researchers show the role of ASIC3 in osteoarthritis: Masashi Izumi, Masahiko Ikeuchi, Qinghui Ji, Toshikazu Tani. Local ASIC3 modulates pain and disease progression in a rat model of osteoarthritis. Journal of Biomedical Science 2012, 19:77 doi:10.1186/1423-0127-19-77.
Highlights: OA was induced via intra-articular mono-iodoacetate (MIA) injection, and pain related behaviors were evaluated including weight bearing measured with an incapacitance tester and paw withdrawal threshold in a von Frey hair test. OA rats showed not only weight-bearing pain but also mechanical hyperalgesia outside the knee joint (secondary hyperalgesia). ASIC3 expression in knee joint afferents was significantly upregulated approximately twofold at Day 14. Continuous intra-articular injections of APETx2 inhibited weight distribution asymmetry and secondary hyperalgesia by attenuating ASIC3 upregulation in knee joint afferents. Histology of ipsilateral knee joint showed APETx2 worked chondroprotectively if administered in the early, but not late phase.

Images: Fast Blue labeling and immunohistochemistry staining for ASIC3 : (a-b) Naïve- model, (c-d) OA-model, (e-f) APETx2 administration to OA-model in early phase. Photos in each row are the same DRG. In (b),(d),(f), large arrows indicate Fast Blue labeled, ASIC3 immunoreactive (ASIC3-ir) DRG cells, while ASIC3-ir cells that were not labeled by Fast Blue are indicated by small arrowheads. More than 100 FB-labeled neurons were analyzed from 4 rats in each group. The percentage of ASIC3-ir knee joint afferents was 18 ± 3% (mean ± SD) in naïve models, 46 ± 4% in OA-models (p = 0.003), and 20 ± 5% in the early-phase APETx2 group (p = 0.006), respectively. Scale bar: 50 μm

Protocol: The [DRG] sections were blocked in 3% normal goat serum for 1 h, then incubated in primary antibody of ASIC3 (Neuromics; Edina, MN, GP 14015, 1:500) overnight in a humid chamber. The next day, the sections were incubated in the secondary antibody (Vector; Burlingame, CA, FI-7000, 1:500, FITC tagged) for 2 h. All antisera used were diluted in PBS containing 1% normal goat serum and 0.05% Triton X-100. Before, between, and after each incubation step, the sections were washed 3 times for 5 min in PBS. Finally, all sections were mounted with Vectashield (Vector, Burlingame, CA).
1. Waldmann R, Champigny G, Bassilana F, Heurteaux C, Lazdunski M: A proton-gated cation channel involved in acid-sensing. Nature 1997, 386:173–177. 2. Lingueglia E: Acid-sensing ion channels in sensory perception. J Biol Chem 2007, 282:17325–17329. note: see for research using our siRNA transfectio reagent for ASIC3 gene expression analysis. 
3. Wemmie JA, Price MP, Welsh MJ: Acid-sensing ion channels: advances, questions and therapeutic opportunities. Trends Neurosci 2006, 29:578–586.
4. Voilley N, de Weille J, Mamet J, Lazdunski M: Nonsteroid anti-inflammatory drugs inhibit both the activity and the inflammation-induced expression of acid-sensing ion channels in nociceptors. J Neurosci 2001, 21:8026–8033.