Optimizing Human Microglia Cultures

Healthy and Happy Cells
Microglia, one of the glial cell types in the CNS, is an important integral component of neuroglia cell network. They act as brain macrophages when programmed cell death occurs during brain development or when the CNS is injured or pathologically damaged.

This makes them an important tool for drug discovery.

We have research proven Human Microglia.

Neuromic's Microglia Cultured in T25 Flask
Related Protocol:

• Place the flask in a clean Biosafety cabinet. 
• Remove the seal on the T-25 Flask. 
• Remove about 15-20ml of media from the flask. Change the cap on the flask, make sure media is not touching the cap, close the cap. 
•  Culture the cells in a 37^oC incubator with 5%CO₂. 
• For best result, do not disturb the culture Flask for at least 72 hours after receiving the T-25 flask the culture has been in. Change the growth medium the next day.

Need Primary Human Cells? Just ask me. pshuster@neuromics.com.

Long Term Culturing of Cells

Non-Perfusion Model
Researchers, for some studies, are demanding the ability to maintain vibrant cultures for long periods of time.

Perfusion models afford this, but are expensive, sensitive and require specialized expertise. Given this, I believed it a good time to represent a protocol that can be used for our primary neurons and can be extrapolated to many of our other primary and stem cells.

Here's the complete protocol.

Rose Ludescher, Manager of Customer Satisfaction, is an expert in helping our customers successfully culturing cells. So if you need help with any of your cell-based assays do not hesitate to contact her. Just email your request to rose@neuromics.com.

Cells from Diseased Donors

Focus on Neuro Diseases

We now have the capability to provide cells of the central and peripheral nervous system from donors with Neuro diseases. These include cells from donors with ALS, AD, PD, and Brain Cancer Donors, to name a few.

We have provided cells to virtually all the large Pharmas and many small and mid-size Bio-techs. We have worked with Novartis to gain 21-CFR compliance for cells that they are using for their eye diseases drug discovery programs.

I am at your “beck and call” should you have interested in exploring specific capabilities further. You can e-mail or call me at 612-801-1007

More FBS Data!

Cells+Media Supplemented with our FBS!

We continue to be pleased with the cell culture images our customers are sharing with us-https://www.neuromics.com/fbs-data

Neurons are notoriously hard to culture. Here're images of Mouse Neurons cultured in media supplemented with our FBS.

Images: Mouse cortical neurons in culture, using Neuromics heat-inactivated FBS (in plating media for two days). Data courtesy of Dr. Saif at Barrow Neurological Institute.

We continue to offer $50 Amazon Gift Card in return for data you share using any of our solutions. Just email the data to Rose Ludescher, Manager of Customer Satisfaction, rose@neuromics.com and she will email you the gift card.

Neuromics' Customers

How They Find Us and What they Buy

Trust is hard to maintain and easily lost. It is important to us that our customers can find us and the products required to meet their research needs.

In order to nurture trust, we follow up with each and every customer to make sure they are happy with their Neuromics' experience. We include the opportunity for them to formally rate this experience @ https://birdeye.com/neuromics-186498936.

We plan on rolling out a new website. Our goal is to make it even easier for customers to find us and present content in a way that aligns with customers' needs. This includes making sure content is configured in a way that works on all platforms used including mobile devices.

As part of the process, we recently surveyed users for areas of research, platforms used, how they find companies like ours and what the purchase. Here're rollups of some of the results.

Our customers most frequently purchase Antibodies and Markers. Cells and Cell-Based Assays are increasingly important, We plan on making it easier for users to find related protocols, publications, data and alternative product options.

Trust, for us, includes the ability for our customers to find the correct solutions and if they prove not to work, offer fixes or full refunds. As in the past, we will be posting updates, publications and customer generated data here.

Need Cells From Diseased Donors?

Just Ask
Some of our customers have expressed the need for Cells that have the phenotype of a given disease. This is especially true for researchers doing drug discovery for neuro-diseases like Alzheimer's and Parkinson's.

Deriving "hard to find" cells from tissue is one of our cornerstones.

If you have need for any type of human cell(s), give us a shot. I can be reached at 612-801-1007 or pshuster@neuromics.com. Thank you. Pete Shuster,CEO and Owner, Neuromics.

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.

Save 100 USD on Our New Cardiomyocytes

Potent, Pure and Easy to Culture

I am pleased to announce the addition of Human Cardiomyocytes to our Stem and Cell Based Assay Solutions.

Image: Human Cardiomyocyte culture.

These cells are optimized to provide addition options for in-vitro testing of drug to drug candidate toxicities allowing researchers to rule out the ineffective and potentially toxic small molecules/compounds early in the process.

I want to make it easy for you to buy and try. If you are not delighted with your results, I will replace free of charge or refund your purchase. Please also note the select positive feedback on our other primary and stem cells:
We recently used the hMSCs derived from Umbilical Cord Blood. Their performance was nothing less than excellent. We were highly impressed with their morphology and their doubling rate. In addition the cells respond very well to accutase and maintain their performance after passaging. We highly recommend this cell line. Rodney Nash, Ph.D, Georgia State University.
Combined Hippocampus, Cortex, and Ventricular Neurons: "I got 10 million cells total after extraction from the tissue. At Day 4 they all developed long axons. Thank you so much for the replacement." Dr. Lidia Gardner, University of Tennessee HSC.
Neuromics always provides excellent products and the best customer service. I highly recommend this company's antibodies and neuronal cultures. Kirsten Raehal, Purdue Pharma

I am always available for product related issues and questions. I cam be reached at direct phone: 612-801-1007 and pshuster@neuromics.com. Pete Shuster, CEO and Owner, Neuromics.

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.

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.

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 NeuroTrak^TM assays enabling kinetic quantification of neurite dynamics (initiation, branching, extension, retraction). NeuroTrack is one of several CellPlayer^TM assays that can be run in IncuCyte Zoom^TM.

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.

Primary Neurons vs PC12 cells for Compound Testing

This publication compares PC12 Cells vs E18 Primary Cortical Neurons. The cells showed permeability to some key compounds where the Neurons did not. This demonstrates the importance of including primary neurons in compound testing assays for Neuro-disease research: Wei Zhang , Radhia Benmohamed, Anthony C. Arvanites, Richard I. Morimoto, Robert J. Ferrante, Donald R. Kirsch, Richard B. Silverman. Cyclohexane 1,3-diones and their inhibition of mutant SOD1-dependent protein aggregation and toxicity in PC12 cells. Bioorganic & Medicinal Chemistry. Elsevier Ltd. All rights reserved.doi:10.1016/j.bmc.2011.11.039.
Abstract: Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by the progressive loss of motor neurons. Currently, there is only one FDA-approved treatment for ALS (riluzole), and that drug only extends life, on average, by 2–3 months. Mutations in Cu/Zn superoxide dismutase (SOD1) are found in familial forms of the disease and have played an important role in the study of ALS pathophysiology. On the basis of their activity in a PC12-G93A-YFP high-throughput screening assay, several bioactive compounds have been identified and classified as cyclohexane-1,3-dione (CHD) derivatives. A concise and efficient synthetic route has been developed to provide diverse CHD analogs. The structural modification of the CHD scaffold led to the discovery of a more potent analog (26) with an EC50 of 700 nM having good pharmacokinetic properties, such as high solubility, low human and mouse metabolic potential, and relatively good plasma stability. It was also found to efficiently penetrate the blood–brain barrier. However, compound 26 did not exhibit any significant life span extension in the ALS mouse model. It was found that, although 26 was active in PC12 cells, it had poor activity in other cell types, including primary cortical neurons, indicating that it can penetrate into the brain, but is not active in neuronal cell potentially due to poor selective cell penetration. Further structural modification of the CHD scaffold was aimed at improving global cell activity as well as maintaining potency. Two new analogs (71 and 73)
were synthesized, which had significantly enhanced cortical neuronal cell permeability, as well as similar
potency to that of 26 in the PC12-G93A assay. These CHD analogs are being investigated further as novel
therapeutic candidates for ALS.

see: Bioorg. Med. Chem. 2011, 19, 613. and J. Med. Chem. 2012, in press

Related Links: Primary Neurons and Astrocytes-Primary human, rat and mouse neurons and astrocytes.

Image: E18 hippocampal neurons stained with Tau (red) and Doublecortin (green). The two proteins overlap in the proximal dendrites (yellow) Axons (low doublecortin content) are red. Blue staining is the nuclear DNA.

Primary Neurons and Cell Based Assays

The feedback I receive from Neuroscientists is consistent. To paraphrase, "gives us healthy, consistent and potent primary cells. I understand the hard work it takes to generate meaningful and publishable results from cell based assays. Our Primary Neurons and Astrocytes are merely inputs for these assays. The real cost is the time invested in culturing and time lost if they don't work.

I have numerous postings on success: Primary Neurons Postings. I wanted to share more data and feedback.

Primary DRGs-Culturing these can be tricky. I make it a point to work with labs to make sure the protocol options best match the desired outcome for assays. This includes replacing cells to make sure we can accurately troubleshoot. This approach insures I can pin point the issues and make sure they are all resolved in round two. Here's a representative testimonial: "Thanks for following up, the DRGs worked great and we were able to get excellent data from them. Thanks so much for working with us." Adam Ross, Dr. Chengji Zhou Lab, UC Davis

Image: DRGs cultured on Calf Skin Collagen.

Primary Hippocampal Neurons-I would like to thank Vimal Swarup, University of Utah for this excellent image.

The cells have been fixed after 48 hrs, they were grown over poly-lysine coated coverslips in the media supplied by Neuromics. Cells were imaged in phase contrast mode with 40x objective.

Put our primary cells to the test!

Potent and Cost Effective Cell Based Assays

I have had many conversations with basic and drug discovery researchers on improving cell based assays. Here's the wish list:

• More potent cells/media
• More accurate analytic tools-quatititative and reproducible results
• Ability to use cells and tools in high throughput/high content screening.
• Cost effectiveness

This wish list is front and center in determining the cells/media and related tools we add to Neuromics' offerings. We are pleased to announce the addition of our Hemogenix's Bioluminomics™ In-Vitro Cell Assays, MSCGro™ Mesenchymal Stem Cell Media and Umbilical Cord Blood derived hMesenchymal Stem Cells.

These provide quantitation, not subjectivity. It includes assay calibration and standardization. It means assay validation. It produces results you can trust and rely on. It means innovation and flexibility. It is advanced technology that is fast to learn, easy to use and above all, cost effective.

Assays options:

• LumiSTEM™ for Primary Stem and Explanted Cells, Stem and Other Cell Lines-Selecting LumiSTEM™ Assays(pdf - 418Kb)
• LUMENESC™ for Mesenchymal Stem/Stromal Cell System-Selecting LUMENESC™ Assays(pdf - 984Kb)
• HALO® for for Human Lympho-Hematopoietic Stem and Progenitor Cell Research.-Selecting HALO™ Assays(pdf - 1.7M)
• Biolumonics Basic Procedures(pdf - 323Kb) 

Available Cells:

Primary Neurons and Astrocytes-Primary human, rat and mouse neurons and astrocytes
STEMEZ^TM Human Neural Progenitor Neuron Discovery Kits-Derived from H9 (WA09) ECSs-Consistent, Easy to Use & Cost Effective
Human Mesenchymal Stem Cells (hMSCs-hMSCs derived from pancreas and umbilical cord blood
Mammalian Cell Lines

Media:
STEMEZ(TM) hN2 Human Neurons Culture Media
MSCGro™ Mesenchymal Stem Cell Media
NbActiv4

I will continue to post customer input and related data on Neuromics' Cell Based Assay Tools.