Osteoblasts Off

I am pleased to announce we now have unlabeled and FITC-labeled Osteoblasts. These are differentiated from our Umbilical Cord Blood derived Human Mesenchymal Stem Cells.

They are designed for:

• Osteogenesis/Bone Formation Studies
• Compound/Small Molecule Testing
• Gene Expression Analysis

Images: (A) Human cord-blood MSCs were expanded in low-serum MSC-Gro^TM to confluence as shown here. (B) were differentiated in osteogenic MSC-Gro^TM. Early stage osteoblasts are shown here; the arrow shows early formation of mineralized matrix. (C)&(D) Mature osteoblasts stained positive for Alizarin red. Phase contrast image at 200 x, scale bar is 50 mmeters.

I will continue to update you on new Cell Based Assay Solutions.

New Near Infrared Apoptosis Detection!

Detecting apoptosis in cells, tissue and living animals

In vitro and in vivo apoptosis detection assays are widely used in basic research and drug discovery. Neuromics has a wide range of kits providing capablilties to meet you unique requirements. We are pleased to offer even more options with our new Far Infrared Kits:
660 Polycaspase in vitro Apoptosis Detection Kit 25-50 Tests $199
660 Caspase-1  in vitro Apoptosis Detection Kit 25-50 Tests $199
NIR FLIVO™ 690 in vivo Apoptosis Tracer Kit 20 Tests-$399
NIR FLIVO™ 747 in vivo Apoptosis Tracer Kit 20 Tests-$399

Seeing is Believing

Images: To demonstrate the capabilities of the NIR-FLIVO® 747 apoptosis tracer, adult wild-type Balb/c mice were either inoculated with HSV-1 virus, which is known to induce apoptosis in the brain, or given a sham treatment. Seven days after viral inoculation, the mice were injected intravenously with either the NIR-FLIVO® 747apoptosis tracer (cat. KF17368), the NIR-FLIVO® 747 free dye (cat. KF17370, DyLight®747), or no reagent. Seven hours after reagent injection, the animals were imaged with a Carestream In-Vivo FX Pro imaging system. Strong caspase activity was located in the brain of the animal treated with HSV-1 and injected with NIR-FLIVO® 747 tracer (cat. KF17367). Minimal signal was detected in the liver region of the HSV-1-treated animal injected with the free dye control (HSV-Infected; NIR747FreeDye+) and of the uninfected mouse injected with NIR-FLIVO®747 tracer; the liver is the route of clearance for FLIVO® tracers. All reagent-injected mice show fluorescence signal in the tail where reagent is likely to pool after IV injection.

Images: Brain abscesses were induced in mice following the intracerebral inoculation of live S. aureus. Animals received intravenous injections of the pan-caspase tracer NIR-FLIVO™ 690 as an apoptosis probe or DyLight®690 (carboxylic acid form) as a control at 17 h post-infection, whereupon signals were acquired 1 h later from brain tissues immediately ex vivo using an IVIS Spectrum (Caliper Life Sciences). The same instrument settings were used to acquire both images. Strong caspase activity, detected with NIR-FLIVO™ 690 (arrows, image on right), was associated with brain abscesses, whereas minimal signal was detected in animals injected with the carboxylic acid control (image on left).

Check them all out
Apoptosis Research Reagents-Detection kits, antibodies and proteins

Everything You Wanted to Know About Stem Cells

Unforgettable way to learn about Stem Cells.

Rap on Dr. Jonathan Garlick Rap on.

If you are doing stem cell related research, check out our:

hNP1™ Human Neural Progenitor & hN2™ Neuron Discovery Kits Derived from H9 (WA09) ECSs-Consistent, Easy to Use & Cost Effective	Neural Stem Cells Media
hMPro™ Human Mesenchymal Progenitors (hMPCs) hESC Derived	Human Mesenchymal Stem Cells (hMSCs) hMSCs Derived from Umbilical Cord Blood
MSCGro™ Mesenchymal Stem Cell Media Proven and Potent Culture Growth and Differentiation Media for Mesenchymal Stem Cells (MSCs)	Cell Cryopreservation Media Maximizes cell recovery, attachment and growth
Expansion/Differentiation Kits SC to Oligodendrocytes and Dopaminergic Neurons Kits	Bioluminomics™ In Vitro Assays Brought to you by HemoGenix®
3-D Cell Based Assay Solutions Nanofibers, Hydrogels and Extracellular Matrix (ECM) Proteins	Stem Cell Research Antibodies
Stem Cell Research Proteins	Rat Neurosphere Neuroprogenitor Tissue Tissue provided live, unseparated, fresh from E18 rat cortex/hippocampus including subventricular zone.

Schwann Cells Markers

Important tool for studying diseases that cause PNS abnormalities or degeneration.

It is a goal of mine to have the best and brightest collection of Neuron-Glial Markers. Here I feature use of one of our Schwann Cell Markers. In this study our p75/NGF antibody is used as a control to determine differences in myelin sheath structure in new born pigs having the Cystic Fibrosis Mutant vs Normal Gene Expression (controls). 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.

Images: CFTR is functionally active in Schwann cells. (A) Primary cultures of porcine Schwann cells were used 4 wk after seeding when they had developed the specific bipolar morphology and a phase-bright cell body under differential interference contrast microscopy. Schwann cells were positive for the phenotypic markers S100 and p75. (B) Whole-cell current recorded in the presence of PKA and ATP in the pipette solution and 1 min after adding 100 μM of CFTR inhibitor GlyH-101 to the bath solution. (Left) Example of currents from one cell; Inset shows voltage-pulse protocol. (Upper Right) Example of current-voltage relationship. (Lower Right) Data from five CFTR+/+ Schwann cells and seven CFTR−/− Schwann cells. *P = 0.003 (Mann–Whitney rank sum test).

Images: Fig. 1. CFTR is expressed in trigeminal nerve Schwann cells. Data are confocal microscopic images of trigeminal nerve immunostained for CFTR (green) and marker indicated above middle panels (red). Nuclei were stained with DAPI (blue). (A) Transverse cross-section with axons stained with β-tubulin III antibody. (B) CFTR−/− trigeminal nerves immunostained for CFTR and β-tubulin III.  (C) Sagittal crosssection immunostained with fluoromyelin. (D) Section stained with S100 antibody, a marker of Schwann cells. (E) Section stained with p75 antibody, also a marker of Schwann cells. (Scale bar, 20 μm.).

I will continue to post new customer pubs and other shared customer data featuring use of our Neuron-Glial Markers.

Epidermal Nerve Fibers in Neuropathic Pain Model

Re-innervation in spared nerve injury (SNI) vs normal rats
Neuromics' Pain and Inflammation Research Antibodies are frequently used to help researchers find root causes of neuropathic pain. This is a fascinating study of re-innervation patterns of epidermal and dermal nerve fibers in a rat neuropathic pain model (Note: our Guinea Pig Polyclonal P2X3 Antibody was used in this study): Liron S. Durakua, Mehdi Hossaini, Barthold N. Schüttenhelm, b, Joan C. Holstege, Martijn Baas, Tom J.H. Ruigrok, Erik T. Walbeehm. Re-innervation patterns by peptidergic Substance-P, non-peptidergic P2X3, and myelinated NF-200 nerve fibers in epidermis and dermis of rats with neuropathic pain. Experimental Neurology Volume 241, March 2013, Pages 13–24. doi.org/10.1016/j.expneurol.2012.11.029.

Non-footpad area vs. footpad.The distribution pattern between the center non-footpad area and footpad is compared for the different subgroups of sensory skin fibers. For epidermal Sub P-IR and upper dermal NF-200-IR fibers there is an equal density of fibers in the center and footpad area suggesting a homogenous distribution over the foot sole of a rat. Whereas for epidermal P2X3-IR fibers there is a significant lower number of P2X3-IR fibers in the footpads as compared to the center non-footpad area, suggesting a heterogeneous distribution in the foot sole for this type of sensory fibers. N = 5 per group. Unpaired t-test. ***: p < 0,001. Scale bar: 250 μm. doi.org/10.1016/j.expneurol.2012.11.029

Illustration of the skin innervation in normal and SNI situation.Illustration showing the innervation pattern of subgroups of sensory skin fibers in the normal situation and in the SNI model 10 weeks PO. In the naïve animals the peptidergic CGRP and non-peptidergic P2X3 fibers have a lower density in the footpads as compared to the non-footpad areas, while the Substance P and NF-200 fibers have an equal distribution over the complete foot sole. In the SNI model there is a significant increase of CGRP epidermal fibers in the medial and lateral area of the foot sole and there is a complete re-innervation of the center area. In addition the footpads in the SNI model are hyper-innervated with CGRP fibers. Substance P and P2X3 and NF-200 fibers show no increased density in the uninjured medial and lateral area after 10 weeks PO. In addition, Substance P and P2X3 fibers hardly re-innervate the center area; however, the NF-200 fibers have the same density of fibers in the denervated area as in the normal situation. In the SNI model the medial, center and lateral area have all an increase in LC's, with the center area being the most prominent one. In addition the epidermis thickness of the SNI model has decreased significantly in all the three areas after 10 weeks PO.
Related Postings: http://neuromics.blogspot.com/search/label/Neuropathic%20Pain.

QCing our hMSC Derived Chondrocytes

We routinely internally test our Human Mesenchymal Stem Cells (hMSCs) and terminally differentiated cells. I would like to share the latest on our hMCS Derived Human Chondrocytes.

Msc derived human chondrocytes in culture-01-2013 from Pete Shuster
We will soon be adding QC and related images for our Osteoblasts and Endothelial Cells.

Stem Cells, Duchenne Muscular Dystrophy (DCM) & Cardiomyopathies

Transplanting Aorta-derived mesoangioblasts (ADMs) to Prevent ADM Related Cardiomyopathy-ADMs can be induced to express cardiac markers, including Nkx2.5, cardiac tropomyosin, cardiac troponin I, and -actinin, and adopt cardiomyocyte morphology. Transplantation of ADMs into the heart of mdx/utrn−/− mice prior to development of DCM prevented onset of cardiomyopathy, as measured by echocardiography, and resulted in significantly higher CD31 expression, consistent with new vessel formation. Dystrophin-positive cardiomyocytes and increased proliferation of endogenous Nestin cardiac stem cells (Neuromics' Chicken Polyclonal Nestin antibody was used as a marker to determine presence of these cells) were detected in ADM-injected heart: JU LAN CHUN, ROBERT O’BRIEN, MIN HO SONG, BLAKE F.WONDRASCH, SUZANNE E. BERRY.Injection of Vessel-Derived Stem Cells Prevents Dilated Cardiomyopathy and Promotes Angiogenesis and Endogenous Cardiac Stem Cell Proliferation in mdx/utrn−/− but Not Aged mdx Mouse Models for Duchenne Muscular Dystrophy. STEM CELLS TRANSLATIONAL MEDICINE 2013;2:000–000. http://dx.doi.org/10.5966/sctm.2012-0107.

Figure 6. Nestin and cardiac myocytes in ADM-transplanted mdx/utrn / heart. Fluorescent microscopy was used to visualize nestin interstitial and striated cells in the heart. (A): There were significantly fewer nestin interstitial stem cells in ADM-injected (dko/ADMs, n 5) mdx/utrn / heart in comparison with age-matched wild-type heart injected with saline (WT/HBSS, n 4). p values were obtained using Student’s t test. No difference was observed between WT/HBSS versus dKO/HBSS (p .0538) or dKO/HBSS versus dKO/ADMs (p .3843). (B–F): Nestin striated cells (green, indicated by white arrows) were observed in four of five mdx/utrn / hearts transplanted with ADMs. (C): A cluster of nestin striated cells (green, indicated by white arrows) and surrounding tissue containing nestin interstitial stem cells (also green, indicated by yellow arrowheads). (D–F): The same cluster of nestin cells (green) shown in (C), at higher magnification, expressed cardiac troponin I (red [E, F]). (G): Some nestin striated cells were also observed in one of four mdx/utrn / hearts injected with saline. (H): Nestin striated cells were not present in saline-injected wild-type heart. Abbreviations: ADM, aorta-derived mesoangioblast; cTpI, cardiac troponin I; DAPI, 4 ,6-diamidino-2-phenylindole; dKO, double knockout mdx/utrn / ; HBSS, Hanks’ balanced saline solution; WT, wild-type.

Conclusion: ADMs delay or prevent development of DCM in dystrophin-deficient heart, but timing of stem cell transplantation may be critical for achieving benefit with cell therapy in DMDcardiac muscle.
Stem Cell Research Reagents.

Shiny, New GF nanoparticle labeled hMSCs

Human Mesenchymal Stem Cells-Green Fluorescent Nanoparticle-labeled-These cells are generated by chemical transfection of CdSe/ZnS nanoparticles, 100 nm in diameter and show a strong fluorescent signal (Excitation maximum= 515 nm; Emission maximum= 520-550 nm) detectable by standard FITC filters.

Image: FITC analysis of cells during fourth passage following transfection. Image obtaining using FITC filter on Olympus CKX41 microscope at 200 X equipped with a Retiga 2000 digital camera and Q-Capture software program. Scale bar is 25 micrometers. Fluorescent nanoparticles are less prevalent within the cytoplasm than earlier passages. Technical Information & Data.

They retain GFNP labeling through 3+ passages and can be differentiation into chondrogenic, adipogenic or osteogenic lineages. Excellent for regeneration, toxicity and related studies. Technical Information & Data.

Image: Osteogenic differentiation of GF nanoparticle-labeled MSCs. Image obtaining using phase contrast Olympus CKX41 microscope at 100 X equipped with a Retiga 2000 digital camera and Q-Capture Pro software program. Scale bar is 25 micrometers. Note the presence of mineral deposits indicating differentiation into osteoblasts. Chondrogenic and adipogenic differentiation was also apparent.

• All Stem Cell Research Reagents.

Pain and the Interplay Between P2Y Receptors with P2X3

P2Y2-P2X3 crosstalk in DRG neurons

Alfredo Ribeiro-da-Silva and his team use our Purinergic Receptor Antibodies to the study role of their expression in Nociceptive and Neuropathic Pain. They referenced their use in 8 publications.

Based on their research, they suspect changes in P2X3 function under pathological conditions are more complex than simple up- or down-regulation of expression at the protein level. This would have profound implications for the dicovery of drugs that target P2X3 expression levels: Gary Mo, Jennifer C. Peleshok, Chang-Qing Cao, Alfredo Ribeiro-da-Silva and Philippe Séguéla. Control of P2X3 channel function by metabotropic P2Y2 UTP receptors in primary sensory neurons. Molecular Pharmacology Fast Forward. Published on December 18, 2012 as doi:10.1124/mol.112.082099.

Images: P2X3 and P2Y2 receptors are co-expressed in rat DRG sensory neurons.(A) DRG sections labeled for P2Y2 (green) and P2X3 (red) and merged (yellow). P2Y2 immunoreactivity is broadly distributed in small-diameter neuronal somata and co-localized with P2X3 (arrows). P2Y2 immunoreactivity can be detected in cells negative for P2X3 (arrow heads). (B) P2Y2 (green) and P2X3 (red) are also colocalized (yellow) in peripheral nerve fibers.

The two receptors were found to be colocalized both in cell bodies and nerve fibers, indicating co-trafficking to peripheral and central terminals. The immunolocalization evidence presented here provides valuable information on the physiological relevance of crosstalks between ionotropic and metabotropic ATP receptors in sensory pathways, yet much work is still needed to fully comprehend the role of nucleotide signaling in pain, especially in pathological conditions. Changes in P2Y2 receptor expression in response to inflammation have been documented (Malin et al., 2008), therefore it will be worth investigating the functional impact of P2Y2-P2X3 interactions on ATP signaling under chronic pain conditions.

I will keep you posted on any new developments.

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.

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.

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. http://dx.doi.org/10.1016/j.neuropharm.2012.11.011. 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. http://dx.doi.org/10.1016/j.jss.2012.11.041
...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. http://dx.doi.org/10.1016/j.pain.2012.03.023.

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. doi.org/10.1016/j.pain.2012.03.023...Sections 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. 

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.

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.

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.

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.

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:

Name	Catalog #	Type	Species	Applications	Size	Price
CNPase	CH23013	Chicken IgY	H; M	ICC; IHC	100 ul	$89
Caspr2	SP15104	Sheep IgG	H; M	IHC; WB; E	100 ug	$365
HSP105	MO20028	Mouse IgG	H; M; R	IHC; WB	100 ul	$155
MAG/Siglec 4a	GT15152	Goat IgG	R	IHC; WB; E	100 ug	$365
MOG	GT15141	Goat IgG	H	IHC; WB; E	100 ug	$365
Mash1	GT15216	Goat IgG	M	IHC; WB; E	100 ug	$365
Mash1	MO15048	Rat IgG	H; M	ICC; WB; E	100 ug	$255
NOGO Receptor	GT15154	Goat IgG	H	IHC; WB; E	100 ug	$365
OMgp	GT15200	Goat IgG	H	WB; E	100 ug	$365
Olig1	RA14141	Rabbit IgG	R	IHC	100 ul 100 ul @ 1mg/ml	$350 $95
Olig1,2,3	MO15059	Mouse IgG	H; R	IHC	100 ug	$305
Olig2	GT15132	Goat IgG	H; M	IHC; WB; E	100 ug	$365
Olig2	RA25081	Rabbit IgG	H; M; R	ICC; IHC; WB; IP	100 ul	$395
Oligodendrocyte Marker O1	MO15001	Mouse IgM	H; M; R	IHC; FC	50 ug	$215
Oligodendrocyte Marker O4	MO15002	Mouse IgM	C; H; M; R	IHC	50 ug	$215
Oligodendrocyte Marker O4-Phycoerythrin Labeled	FC15013	Mouse IgM	H	FC	100 Tests	$305
PDGF R Alpha/CD140A	GT15150	Goat IgG	M	IHC; WB; E	100 ug	$365

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.

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:

Name	Catalog #	Type	Species	Applications	Size	Price
Native Human Chondrocytes-Pilot	SC00A7-100K	Primary Cells	H	Cell Assays	100,000 Cells	$245
Native Human Chondrocytes-HCS	SC00A7-500K	Primary Cells	H	Cell Assays	500,000 Cells	$449
Native Human Chondrocytes-HTS Pilot	SC00A7-4000K	Primary Cells	H	Cell Assays	4X1,000,000 Cells	$2,399
Native Human Chondrocytes-HTS	SC00A7-1000K	Primary Cells	H	Cell Assays	1,000,000 Cells	$799
Fluorescein Labeled Human Chondrocytes-Pilot	SC00A8-100K	Primary Cells	H	Cell Assays	100,000 Cells	$295
Fluorescein Labeled Human Chondrocytes-HCS	SC00A8-500K	Primary Cells	H	Cell Assays	500,000 Cells	$449
Fluorescein Labeled Human Chondrocytes-HTS Pilot	SC00A7-1000K	Primary Cells	H	Cell Assays	1,000,000 Cells	$799
Fluorescein Labeled Human Chondrocytes-HTS	SC00A7-4000K	Primary Cells	H	Cell Assays	4X1,000,000 Cells	$2,399
Rhodamine Labeled Human Chondrocytes-Pilot	SC00A9-100K	Primary Cells	H	Cell Assays	100,000 Cells	$295
Rhodamine Human Chondrocytes-HCS	SC00A9-500K	Primary Cells	H	Cell Assays	500,000 Cells	$449
Rhodamine Labeled Human Chondrocytes-HTS Pilot	SC00A9-1000K	Primary Cells	H	Cell Assays	1,000,000 Cells	$799
Rhodamine Labeled Human Chondrocytes-HTS	SC00A9-4000K	Primary Cells	H	Cell Assays	4X1,000,000 Cells	$2,399
Human Chondrocytes Media	SC00PC3-100	Cell Growth Media	H		100 ml 500 ml	$79 $199

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.