Tuesday, June 28, 2016

Cells Detachment

Easy, Gentle and Complete

I often get asked if trypsin is the best reagent to detach primary and stem cells. The problem with a trypsin is that it could lyse a percentage of the cells.

We recommend our DetachinTM. It offers the following benefits:
  • Gentle and rapid detachment.
  • Maximum cell viability.
  • Effective on a wide range of cells.
  • No mammalian or bacterial byproducts.
  • Stable at 4°C for 2 months.
  • No need to wash detached cells.
Here's a recent publication referencing the use of Detachin: Yu Hou, Qi Feng, Miao Xu, Guo-sheng Li, Xue-na Liu, Zi Sheng, Hai Zhou, Ji Ma, Yu Wei,1 Yuan-xin Sun,Ying-yi Yu, Ji-hua Qiu, Lin-lin Shao, Xin-guang Liu, Ming Hou, and Jun Peng. High-dose dexamethasone corrects impaired myeloid-derived suppressor cell function via Ets1 in immune thrombocytopenia. Blood 2016 :blood-2015-10-674531; doi:10.1182/blood-2015-10-674531 ...Both PBMCs and splenocytes were cultured in complete medium for 7 days. Each culture was supplemented with recombinant human IL-6 (10 ng/mL; R&D Systems, Minneapolis, MN) and granulocyte macrophage-colony-stimulating factor (10 ng/mL; R&D Systems) in the presence or absence of DXM and incubatedin humidified air with 5%CO2 at 37°C. Cells cultured in medium alone were runin parallel as controls. Adherent cells were removed using a nonprotease cell detachment solution Detachin (Neuromics, Edina, MN). CD331 cells were isolated by anti-CD33 magnetic microbeads and LS column separation (Miltenyi Biotec, Bergisch Gladbach, Germany), per manufacturer's instructions. The purity of the isolated cells was found to be higher than 90% by flow cytometry

Figure 1. The number of MDSCs and their expression of Arg-1/iNOS in the peripheral blood. (A) The representative scatter-gram of CD11b1CD331HLA-DRlow cells within the gate of PBMCs. Histograms of Arg-1 and iNOS in CD11b1CD331HLA-DRlow cells from healthy control patients and patients with ITP before any treatment was initiated. (B) The percentage of CD11b1CD331HLA-DRlow cells in hemolyzed whole blood from primary active patients with ITP (n 5 21) and healthy control patients (n 5 18). (C-D) The expression (mean fluorescence intensity) of Arg-1 (C) and iNOS (D) in circulating MDSCs compared between patients with ITP (n 5 21) and control patients (n 5 18). Significance between the 2 groups was determined by Student-Newman-Keuls test.
We are working hard to find new ways for you to optimize your cell based assays.

Cells Detachment

Easy, Gentle and Complete

I often get asked if trypsin is the best reagent to detach primary and stem cells. The problem with a trypsin is that it could lyse a percentage of the cells.

We recommend our DetachinTM. It offers the following benefits:
  • Gentle and rapid detachment.
  • Maximum cell viability.
  • Effective on a wide range of cells.
  • No mammalian or bacterial byproducts.
  • Stable at 4°C for 2 months.
  • No need to wash detached cells.
Here's a recent publication referencing the use of Detachin: Yu Hou, Qi Feng, Miao Xu, Guo-sheng Li, Xue-na Liu, Zi Sheng, Hai Zhou, Ji Ma, Yu Wei,1 Yuan-xin Sun,Ying-yi Yu, Ji-hua Qiu, Lin-lin Shao, Xin-guang Liu, Ming Hou, and Jun Peng. High-dose dexamethasone corrects impaired myeloid-derived suppressor cell function via Ets1 in immune thrombocytopenia. Blood 2016 :blood-2015-10-674531; doi:10.1182/blood-2015-10-674531 ...Both PBMCs and splenocytes were cultured in complete medium for 7 days. Each culture was supplemented with recombinant human IL-6 (10 ng/mL; R&D Systems, Minneapolis, MN) and granulocyte macrophage-colony-stimulating factor (10 ng/mL; R&D Systems) in the presence or absence of DXM and incubatedin humidified air with 5%CO2 at 37°C. Cells cultured in medium alone were runin parallel as controls. Adherent cells were removed using a nonprotease cell detachment solution Detachin (Neuromics, Edina, MN). CD331 cells were isolated by anti-CD33 magnetic microbeads and LS column separation (Miltenyi Biotec, Bergisch Gladbach, Germany), per manufacturer's instructions. The purity of the isolated cells was found to be higher than 90% by flow cytometry

Figure 1. The number of MDSCs and their expression of Arg-1/iNOS in the peripheral blood. (A) The representative scatter-gram of CD11b1CD331HLA-DRlow cells within the gate of PBMCs. Histograms of Arg-1 and iNOS in CD11b1CD331HLA-DRlow cells from healthy control patients and patients with ITP before any treatment was initiated. (B) The percentage of CD11b1CD331HLA-DRlow cells in hemolyzed whole blood from primary active patients with ITP (n 5 21) and healthy control patients (n 5 18). (C-D) The expression (mean fluorescence intensity) of Arg-1 (C) and iNOS (D) in circulating MDSCs compared between patients with ITP (n 5 21) and control patients (n 5 18). Significance between the 2 groups was determined by Student-Newman-Keuls test.
We are working hard to find new ways for you to optimize your cell based assays.

Monday, June 20, 2016

MeCP2 and Nerve Injury

i-Fect used In Vivo for Study

Researchers use our i-FectTM to effectively deliver miR-126 in vivo to modulate Methyl-CpG-binding protein 2 (MeCP2).

MeCP2 regulates gene expression through activation, repression and chromatin remodeling. Mutations in MeCP2 cause Rett syndrome, and these patients display impaired nociception. The researchers observed an increase in MeCP2 expression in mouse dorsal root ganglia (DRG) after peripheral nerve injury: Melissa T. Manners, Adam Ertel, Yuzhen Tian and Seena K. Ajit. Genome-wide redistribution of MeCP2 in dorsal root ganglia after peripheral nerve injury. Epigenetics & Chromatin 20169:23. DOI: 10.1186/s13072-016-0073-5© The Author(s) 2016 Received: 11 March 2016. Accepted: 27 May 2016Published: 7 June 2016...miRNA administration protocol was adapted from previous report of intrathecal miRNA delivery. To administer miRNA mimics, a polyurethane catheter (25G, 5.5 cm long, SAI infusion) was placed into the intrathecal space of the lumber L4–L5 vertebrae under isoflurane anesthesia. The catheter was stereotactically secured under the skin and occluded between injections. A custom miRCURY (Exiqon) miR-126 mimic containing a 5′ cholesterol tag and 3′ fluorescein label was injected at 2 nmol concentration with 4 µl iFECT transfection reagent (Neuromics). A total of 6 µl was delivered into the catheter connection juncture using a 25G blunt end needle on a Hamilton syringe. The catheter was then flushed with 7 µl sterile PBS to ensure miRNA reached the intrathecal space...

Figure: Expression of miR-126 and its target genes Dnmt1 and Vegfa in the DRG after nerve injury. a Relative expression of miR-126 determined by qPCR shows a reduction in miR-126 in SNI model compared to DRG from sham control. U6 was used for normalization (n = 8 sham, n = 7 SNI). b Relative expression of Dnmt1 mRNA and c Vegfa transcripts showed an increase in the DRG after nerve injury compared to control (n = 3). Gapdh was used as a normalizer. d Representative Western blot and quantification showed an increase of Dnmt1 protein in the DRG after nerve injury. e Western blot and quantification showed Vegfa protein was not significantly different in DRG after nerve injury (n = 3 from pooled samples, three DRG were pooled for each sample).


Conclusions: The study shows a regulatory role for MeCP2 in that changes in global redistribution can result in direct and indirect modulation of gene expression in the DRG. Alterations in genome-wide binding of MeCP2 therefore provide a molecular basis for a better understanding of epigenetic regulation-induced molecular changes underlying nerve injury.

Sunday, June 12, 2016

Neuroscience Cell Based Markers Applications

Progenitors, Neuronal, Astroglia and PNS Markers A to Z
Our Neuron/Synapse, Astrocytes, Glia, Microglia, Olidogodendrocytes, Progenitors and Schwann Cell Markers continue to widely used and frequently published.
Here're some recent examples: Cinzia Ambrosi , Cynthia Ren, Gaelle Spagnol, Gabriel Cavin, Angela Cone, Elena E. Grintsevich, Gina E. Sosinsky, Paul L. Sorgen. Connexin43 Forms Supramolecular Complexes through Non-Overlapping Binding Sites for Drebrin, Tubulin, and ZO-1. Published: June 9, 2016. http://dx.doi.org/10.1371/journal.pone.0157073...chicken anti-GFAP in blue (Neuromics, Catalog # CH22102)...
Figure 1. Cx43 and drebrin colocalization analysis in brain and cellular models. (A) Rat brain transversal slice mosaic shown after multiple immunolabeling with antibodies anti-Cx43 (red), anti-drebrin (green), and anti-GFAP (blue) as astrocytes marker. White boxes localize the area enlarged in insets 1, 2, and 3 (six fold enlargement). Colocalization of drebrin and Cx43 (yellow) is especially noticeable around the blood vessels (inset 2) and in regions rich of astrocytes (insets 1 and 3). The different regions of the brain were labeled. Cultured astrocytes (B and C) and Vero cells (D and E) were immunolabeled with anti-Cx43 (red), anti-drebrin (green), and anti-actin (blue). White arrows indicate zones of colocalization of Cx43, drebrin and actin that were enlarged in the insets (white boxes, three fold enlargement).

Xiangchen Li, Yu Guo, Yaxin Yao, Jinlian Hua, Yuehui Ma, Changqing Liu, Weijun Guan.Reversine Increases the Plasticity of Long-Term Cryopreserved Fibroblasts to Multipotent Progenitor Cells through Activation of Oct4. International Journal of Biological Sciences 2016; 12(1): 53-62. doi: 10.7150/ijbs.12199...anti-GFAP and NSE (1:200,Neuromics, MN, USA)...
Save 70 USD on High Titer Neuron/Synapse Markers-Only 225/100 ul (Through June 30, 2016).
We will continue to post news on our Neuroscience Research Solutions!

Friday, June 03, 2016

Human Astroglia and Schwann Cells-BBB Model

Broadening our Capabilities

At the core of our solutions are many options for primary human cells. We are especially pleased that we have growing capabilities to provide new cells to researchers studying autoimmune neuro-degenerative diseases like ALS and MS with the addition of:
Schwann Cells
Human Schwann Cells (HSwC) are isolated from human spinal nerve. HSwC are cryopreserved at passage one and delivered frozen. Each vial contains >5 x 10^5 cells in 1 ml volume. HSwC are characterized by immunofluorescence with antibodies specific to S100, GFAP, and CD90. HSwC are negative for HIV-1, HBV, HCV, mycoplasma, bacteria, yeast, and fungi. HSwC can to further expand for 10 population doublings in our Schwann Growth medium (cat # SGM001).
Human Astrocytes
 Human Brain Astrocytes cultured with AlphaBioCoat.
Human Blood Brain Barrier Model
I will continue to post updates here.

Wednesday, May 18, 2016

IGF and Mood

Gender Specific?

IGF is a known mood modulator. This paper hypothesizes that the impact of increased IGF is sex dependent: Victor Munive, Andrea Santi and Ignacio Torres-Aleman. A Concerted Action Of Estradiol And Insulin Like Growth Factor I Underlies Sex Differences In Mood Regulation By Exercise. Scientific Reports 6, Article number: 25969 (2016) doi:10.1038/srep25969.

Our IGF-I R/CD221-Fluorescein Labeled was used to link Estrodial with elevating IGF-I levels in female mice.
Images: (A) Cultured brain endothelial cells accumulate significantly more IGF-I after treatment with 10−10M estradiol (E2). Representative blot is shown (*p less than 0.05 vs control; n = 8 per group). (B) The effects of E2 were mediated by α E2 receptors as only the α inhibitor MPP blocked E2 actions. Representative blots are shown (*p less than 0.05; n = 8). (C) Levels of IGF-I receptor (green) at the cell membrane of endothelial cells are markedly increased three hours after addition of 10−10M E2.

Summary: Sex-specific responses to physiological neuroprotective stimuli such as physical activity, that modulate mood in part through modulation of endocrine signals, contribute to sex differences in mood homeostasis. Hopefully, a better understanding of these differences will help us gain insight of sex differences in the incidence of mood disorders.

Friday, May 13, 2016

Cancer-Associated Fibroblasts and Cancer Cell Migration

Highway for Cancer Metastasis

Cancer Associated Fibroblasts provide the Matrices  (ECMs) serve as cancer’s assistants as it spreads throughout the body.



Cancer cells (red) migrate on a CAF-derived extracellular matrix (green). VANDERBILT UNIVERSITY; BEGUM ERDOGAN, DONNA WEBB

Unlike other ECMs, which normally form dense meshes, these arrange into parallel bundles forming the highway for tumor cell migration (The Scientist-December 15, 2015).

Monday, May 02, 2016

New Neuronal Astroglial PNS Markers

Save 70 USD on High Titer Antibodies
The foundation of our company is built on our catalog of proven, published and high titer Neuron-Astroglia and PNS Markers. We are pleased to offer 75 USD off our latest additions.
Antibody
Type
Species Reactivity
Applications
Mouse IgG
Chicken IgY
Chicken IgY
Mouse IgG
Mouse IgG
Mouse IgG
Mouse IgG
Mouse IgG
Chicken IgY
Rabbit IgG
Mouse IgG
B; H; M; R
B; H; M; R
H; M; R
B; H; M; R
B; H; M; R
H; R
Ch; H; M; Pr; R
Ch; H; M; R
H; M; R
H; M; R
B; H; M; R
IF; WB
ICC; IF; IHC; WB
ICC; IF; IHC; WB
IF; WB
IF; WB
IF; WB
ICC;
IHC; WB
ICC; IHC; WB
ICC; IF; IHC; WB
ICC; IF; IHC; WB

Images: Left: View of mixed neuron/glial cultures stained with Aldolase-C (green) and our rabbit antibody to NeuN/FOX3 (red). MCA-4A9 antibody reveals strong cytoplasmic staining in astrocytes, while Rabbit Fox3/NeuN antibody shows nuclear and distal cytoplasmic staining in neuron cells and is complete absence of astrocytes. Blue is a DNA stain. Middle and Right: Mouse brain sections (fixed by transcardial perfusion with 4% paraformaldehyde) stained with Aldolase-C (red) and our chicken Vimentin antibody (green). In the striatum (Middle), Aldolase-C positive astrocytes are highly co-stained Vimentin, which results in yellow to gold colors. In the cerebellum (Right), however, Aldolase-C positive Purkinje cells do not express vimentin, which results in red color. Insets show a higher magnification picture of MCA-4A9 single labeling in red. Nuclei are labeled with DAPI (blue).
Our clients often use these antibodies for double and triple labeling like the above example.


We plan on aggressively be adding more and more of these type of antibodies. Stay tuned.

Tuesday, April 26, 2016

ELISA Blocking Buffers

Paramount to Reducing Background Noise and Attaining an Accurate Signal

Once the antibody or protein antigens have been properly adsorbed onto the ELISA/EIA plate, the next critical step in creating a reliable immunoassay is the blocking of the plate. This key is:

  • Preventing nonspecific binding 
  • Reducing ELISA background signal 
  • Blocking nonspecific binding to adsorbed proteins 
  • Stabilizing proteins adsorbed to plate for better interactions 



Neuromics's ELISA Blocking Buffer formulations reduce nonspecific binding of sample and assay components to the ELISA well while stabilizing the coated protein. Six formulations offer significant benefits to different assay situations. Browse the product selections below, or order our Blocking Buffer Optimization Pack to try three formulations at an economical rate.

Friday, April 15, 2016

Isolating and Selecting T-Cells

Save 100 USD on New Kits

We are pleased to announce the addition of our New T-Cell Isolation/Selection Kits. They are fast and easy. Check out the Short Protocol.

Figure CD4+ T-Cell Isolation Process doi:10.1371/journal.pone.0035798.

We also have T-Cell Expansion Kits!
NameCatalog #TypeSpeciesApplicationsSizePrice
StrepMan Magnet for 15ml and 50ml Tubes6-5650-065Cell Isolation-SeparationHCell Assays1 Magnet$400
CD3 Fab Streptamer Isolation Kit MB6-8000-201Cell Isolation-SeparationH1 Kit$699
CD4 Fab Streptamer Isolation Kit MB6-8000-206Cell Isolation-SeparationH1 Kit$699
CD8 Fab Streptamer Isolation Kit MB6-8000-203Cell Isolation-SeparationH1 Kit$699
CD25 Fab Streptamer Isolation Kit MB6-8000-207Cell Isolation-SeparationH1 Kit$699
We'll be adding more and more kits in the coming months and will keep you posted.

Saturday, April 02, 2016

Magic Red and Cancer Research

Measure Apoptosis in Real Time in Cancer Cells

Magic Red™ (MR) Kits measure apoptosis via active caspases and cathepsins in whole living, intact cells - no lysis required. As apoptosis progresses and caspase activity increases, the red fluorescent signal increases.

In this example, Magic Red is used to measure apoptosis in HeLa Cells after treatment with Artesunate (ART), an anti-malarial drug and potential chemotherapy agent.



Figure: ART activates lysosomal function. A, HeLa cells were first treated with ART (50 μM) for 12 h, followed by LTR labeling for 30 min, and further analyzed using confocal microscopy. B, HeLa cells were loaded with LTR following time course drug treatment, and fluorescence intensity of 10,000 cells/sample was measured by flow cytometry. C, HeLa cells were treated as indicated in A and then incubated with Magic Red cathepsin L and LTG for 30 min and observed under a confocal microscope. D, HepG2 cells were treated as in A and then labeled with LTR described earlier in A. E, HepG2 cells were treated as in A, followed by a Magic Red cathepsin B and L assay for 45 min. Fluorescence intensity of 10,000 cells/sample was measured using flow cytometry. F, HeLa cells were first loaded with DQ Red BSA for 1 h and then treated with ART (50 μM) for 18 h and incubated with LTG for 30 min. G, HeLa cells were treated with ART for 18 h and then immunostained for LAMP1. H, LAMP1 signals of 80 cells from three independent experiments were quantified by ImageJ (Student's t test, #, no significance). I, HeLa cells were treated with ART for the indicated time, and then Western blot was performed to detect the LAMP1 protein level. Scale bar, 10 μm. Error bars, S.D. doi: 10.1074/jbc.M114.564567

We have a variety of Apoptosis Kits for testing potential Chemotherapeutic Agents.

Thursday, March 31, 2016

Human Astrocytes

Cortex Derived Astrocytes

Neuromics is pleased to be offering yet another option for culturing Human Astrocytes. They can be passaged up to 10X-Only 749 USD/500,000 Cells

They are deigned to be easy to culture and grow.

 Image: Human Brain Astrocytes cultured with our AlphaBioCoat.
Check out our large offering of Neurons, Astroglia, Progenitors, Brain Endothelial Cells/Pericytes and Blood Brain Barrier (BBB) Model. Should you have questions on these or any of our offerings, I can be reached directly at 612-801-1007 or pshuster@neuromics.com. Thank you, Pete Shuster-CEO and Owner.

Thursday, March 24, 2016

Epigenetics and Pain Research

i-Fect Used to Study Impacts

Our i-Fect siRNA, miRNA and shRNA Trasfection Kit was recently used to study the impact of G9a-specific siRNA (AGUAACGGGCAUCAAUGC) on Mu Opioid Receptors: Yuhao Zhang, Shao-Rui Chen, Geoffroy Laumet, Hong Chen and Hui-Lin Pan. Nerve Injury Diminishes Opioid Analgesia through Lysine Methyltransferase-Mediated Transcriptional Repression of µ-Opioid Receptors in Primary Sensory Neurons. First Published on February 25, 2016, doi: 10.1074/jbc.M115.711812... In some SNL rats, G9a-specific siRNA (4 µg) or the negative control siRNA was administered intrathecally. G9a-specific siRNA(AGUAACGGGCAUCAAUGC) or universal negative control siRNA (#SIC001, Sigma-Aldrich) was mixed with i-Fect (Neuromics, Edina, MN) to a final concentration of 400 mg/L for the intrathecal injections...

Figures: G9a knockdown with siRNA reverses the MOR expression in the DRG and the morphine analgesic effect diminished by nerve injury. (A,B) Quantitative PCR (A) and Western blotting (B) analyses show the mRNA and protein levels of MORs in the DRGs of sham and SNL rats treated with control or G9a-specific siRNA (n = 10 rats in each group). The ipsilateral L5 and L6 DRG tissues were removed 24 h after the last siRNA injection. The amount of MOR mRNA and protein was normalized to GAPDH in the same samples, and the mean value of MOR levels in sham control rats was considered to be 1. (C) Time course of the intrathecal morphine effects on the tactile and pressure withdrawal thresholds in sham and SNL rats treated with G9a-specific siRNA or negative control siRNA (n = 9 rats in each group). The withdrawal thresholds after the last siRNA injection were plotted as the baseline control (BL).

Summary: The findings provide new insight into the epigenetic mechanism regulating MOR expression in primary sensory neurons in neuropathic pain. This multidisciplinary approach provides conclusive evidence for G9a as a key chromatin regulator responsible for MOR downregulation in the DRG and the analgesic efficacy of opioids reduced by nerve injury. A better understanding of the epigenetic mechanisms underlying nerve injury-induced downregulation of MORs in primary sensory neurons could help improve the analgesic efficacy of opioids for treating chronic neuropathic pain. G9a inhibitors could be used to enhance the opioid analgesic effect and reduce opioid consumption in patients with chronic neuropathic pain.

Tuesday, March 15, 2016

3-D Tissue Models Made Easy

3 Steps to an in vivo Like Assay
We provide both CollaGel Hydrogels with different stiffness options and ready made 3-D Human Tissue Models.

 Building these models can be done in 3 easy steps:
  • Step 1: Thaw our CollaGel Hydrogel solution 
  • Step 2: Add your cell suspensions 
  • Step 3: Watch it gel
HUVECS intergrated into 3-D CollaGel environment.

Due to the way in which it gels, our Collagel Hydrogel is well suited to Bio Printing models.

I will be posting more data as it becomes available.

Saturday, March 12, 2016

CRISPR Everywhere

Nature Explores What it Means

Here's the link: http://www.nature.com/news/crispr-everywhere-1.19511
We are part of the CRISPR everywhere revolution!

Features Include:

  • Simple-"Cut and Paste" Integration and Editing. 
  • Check out our Sleeping Beauty Transposon System Users Manual 
  • Potent-100% Stable with No Off target effects with the safest transposon insertion profile. 
  • Fast- Transfect with our transposase + Sleeping Beauty TransposonTM Reporter Series Vectors. 
  • Cost Effective- Starting at $329

Friday, March 04, 2016

Check Out Our 3D Human Skin Tissue Model

In vivo Like Model for Studying Skin Diseases/Irritants

I am pleased to announce this new addition to our Cell Based Assay offerings.

3D Human Skin Tissue Model
Cross section of Human Skin Model
The model includes precultured tissue in our engineered and tissue/cell specific Collagel-Hydrogels.

Our 3D human skin model is manufactured in a GMP lab. The model is engineered from multilayered, differentiated epidermal cells. The tissue is provided cultured in inserts using serum free media. The model mimics human skin. It provides an in vivo like environment for studying dermal related diseases and cancers. It is also excellent for skin toxicology and irritancy assays.

Check all our  3D Models:
Name Applications Size Price
Human Blood Brain Barrier Model In vivo Like Assays 6 wells
12 well
24 well
$1,780
$2,600
$3,625
3D HUVEC Model In vivo Like Assays 6 well
12 well
24 Well
$1,269
$1,399
$1,539
3-D Human Skin Tissue Model In vivo Like Assays 6 well
12 well
24 Well
$1,269
$1,399
$1,539

Questions? Contact me directly-Pete Shuster, CEO and Owner, 612-801-1007 or pshuster@neuromics.com.

Monday, February 22, 2016

Stem Cell "Stemness" and Differentiation States

Markers Play a Key Role

Our Stem Cell Markers are widely used and frequently published. These play an important role for understanding the molecular events that take place during the differentiation of human pluripotent cells. They are essential for the protocols aimed at insuring the generation high of quality differentiated cells.

Here's an excellent study on the determining the state of differentiating stem cells: Julio Castaño, Cristina Morera, Borja Sesé, Stephanie Boue, Carles Bonet-Costa, Merce Martí, Alicia Roque, Albert Jordan, Maria J. Barrero. SETD7 Regulates the Differentiation of Human Embryonic Stem Cells. Published: February 18, 2016DOI: 10.1371/journal.pone.0149502...SOX2 (Neuromics GT15098)...

Figure: SETD7 is expressed at very low levels in pluripotent human cells and induced during differentiation. (A) Average rank of the top 700 most differentially expressed genes between pluripotent (iPSCs or ESCs) and fibroblasts, including those upregulated in pluripotent cells (left panel) and upregulated in fibroblasts (right panel). (B) SETD7 mRNA levels in human ESCs grown under self-renewal conditions (UndES[4]), in vitro differentiated human ESCs (DifES[4]), human fibroblasts (HFF), two lines of human keratinocytes (HEK1 and HEK2) and two lines of iPSCs generated from keratinocytes ([H]KiPS4F and KiPS4F1). Mean and standard deviation of three technical replicates is shown. Induction of SETD7 mRNA levels during ES[4] differentiation was confirmed in more than four independent differentiation experiments. (C) Western blot showing SETD7 protein levels in pluripotent and somatic cells. Loading control beta actin (ACTB) is also shown. (D) Western blot showing protein levels of SETD7, AFP, OCT4 and SOX2 in under self-renewing conditions and in vitro differentiated human ESCs. Loading control alpha tubulin (TUBA) is also shown. One representative experiment out of three is shown. (E) Genomic visualization of the levels of H3K72me3, H3K4me3, H3K4me2, H3K36me3 and RNA polymerase II (Pol II) in the human embryonic stem cell line H1 around the SETD7 gene according to ENCODE. A non-methylated CpG island is depicted in green. (F) Levels of H3K4me2 and H3K27me3 at SETD7 gene promoter region (27 bp upstream of the transcription start site) in pluripotent and somatic cells determined by chromatin immunoprecipitation (ChIP) and ploted relative to the input. IgGs wer used as negative control. Bars show the mean and standard deviation of three independent immunoprecipitations...doi:10.1371/journal.pone.0149502.g001

Neuromics is pleased to provide many different stem cell related options for your cell based assays. I will continue to post updates.

Monday, February 15, 2016

3-D Cell Based Assays Are Evolving

3-D Multicell Models and Gels

There is a growing body of publications of why 3-D matters in drug discovery and toxicology assays. This table shows important distinctions for single cell assays.
Cellular characteristics 2D 3D Refs.
Morphology Sheet-like flat and stretched cells in monolayer Natural shape in spheroid/aggregate structures 20,24,50
Proliferation Often proliferate at a faster rate than in vivo May proliferate at a faster/slower rate compared to 2D-cultured cells depending on cell type and/or type of 3D model system 17,51
Exposure to medium/drugs Cells in monolayer are equally exposed to nutrients/growth factors/drugs that are distributed in growth medium Nutrients and growth factors or drugs may not be able to fully penetrate the spheroid, reaching cells near the core 24,52
Stage of cell cycle More cells are likely to be in the same stage of cell cycle due to being equally exposed to medium Spheroids contain proliferating, quiescent, hypoxic and necrotic cells 18,24,53
Gene/protein expression Often display differential gene and protein expression levels compared to in vivo models Cells often exhibit gene/protein expression profiles more similar to those in vivo tissue origins 17,40,54
Drug sensitivity Cells often succumb to treatment and drugs appear to be very effective Cells are often more resistant to treatment compared to those in 2D culture system, often being better predictors of in vivo drug responses 17,33
Table: Key Differences in Cellular Characteristics and Processes in Two-Dimensional and Three-Dimensional Culture Systems

From the table, we see, for the most part, the overall advantages of cells in 3-D for generating more in vivo like data. We also see potential issues like: "Nutrients and growth factors or drugs may not be able to fully penetrate the spheroid, reaching cells near the core".  

We have resolved penetration issues with our engineered Collagel Hydrogels. We have further enhanced the assays by including multicell assays that even more closely mimic the in vivo environment.

Our Blood Brain Barrier (BBB) Model, for example, includes our:
These cells are pre-cultured in the engineered gels and cost a fraction of the individual components (see: culturing and assay protocol).
Name Catalog #

Size Price
Human Blood Brain Barrier Model 3D45002-6 6 wells
12 well
24 well
$1,780
$2,600
$3,625
Our intention is for you to generate the most in vivo like data possible.