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 Detachin^TM. 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 Detachin^TM. 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.

MeCP2 and Nerve Injury

i-Fect used In Vivo for Study

Researchers use our i-Fect^TM 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.

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!

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.