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Primary neuron culture

Neuvitro Corporation owns the leading technology to provide professional coating with the broadest coverage of substrates, such as PDL, PLL, PLO, Collagen, Gelatin, Laminin, Fibronectin, PDL+Laminin, PDL + Fibronectin, PLO+Laminin, PLO+Fibronectin, PDL+Laminin+Fibronectin, etc. The majority of coated German coverslips available from Electron Microscopy Science, ThermoFisher Scientific, and VWR is actually manufactured by Neuvitro Corporation. Free samples are available.

Primary mouse hippocampal neuron culture on Neuvitro coated German glass coverslips

List of German glass coverslips coated for primary neuron culture

 

Researchers use Neuvitro products for neuron and cell culture:

Science
April 30, 2015,
Research Article
Reversible centriole depletion with an inhibitor of Polo-like kinase 4

 

BMC Immunology, 

May 12, 2015
Research Article
Targeting epidermal fatty acid binding protein for treatment of experimental autoimmune encephalomyelitis

 

Journal of Cell Biology
2014, 206: 923-936
Research Article
A matrix metallopreteinase mediates long-distance attenuation of stem cell proliferation

 

Nature
Article #6211, doi: 10.1038, Sept 5 2014

Optical assay of erythrocyte function in banked blood

 

Nature
May 15, 2015,
Scientific report
Number of nanoparticles per cell through a spectrophotometric method- A key parameter to assess nanoparticle-based cellular assays

 

The Journal of Biological Chemistry
Feb. 6, 2015
Research Article

4E-BPs control fat storage by regulating the expresion of Egr1 and ATGL

 

EMBO-Molecular Systems Biology
2014, 206: 923-936
Research Article
Systems-wide analysis of BCR signalosomes and downstream phosphorylation and ubiquitylation

 

The Journal of Clinical Investigation
May 26, 2015
Research Article
Microglia regulate blood clearance in subarachnoid hemorrhage by heme oxygenase 1

 

Molecular Biology of The Cell
June 30, 2014
Research Article

FUS is sequestered in nuclear aggregates in ALS patient fibroblasts

Journal of Biomedical Materials Research

 

The Journal of Clinical Investigation
April 27 , 2015,
Research Article
Microglia regulate blood clearance in subarachnoid hemorrhage by heme oxygenase-1

 

Symbiosis
May 20, 2015,
Research Article

Topiramete protects pericytes from glucotoxicity: Rle for mitochondrial CA VA in cerebromicrovascular disease in diabetes

 

Jan 28, 2015
Special report
Adhesion and activation of Platelets from subjects with coronary artery disease and apparently healthy individuals on biomaterials

 

Molecular Cellular Biology
doi: 10.1128 / MCB.01630-13
Research Article
Activation of stress response pathways promote formation of antiviral granules and restrct virus replication

 

Journal of Cell Biology
2014, 204: 559-573
Research Article
Aggregation state determines the localization and function of M1-and M23-aquaporin-4 in astrocytes


Biotechniques

November, 2014

Report
Marking cells with infrared fluorescent proteins to preserve photoresponsiveness in the retina

 

Methods
Volume 66, Issue 3, 1 April 2014, Pages 370–379
Developmental Biology
Preparation of developing Xenopus muscle for sarcomeric protein localization by high-resolution imaging


Journal of Marine Biology
Volume 2014 (2014), Article ID 769356, 11 pages
http://dx.doi.org/10.1155/2014/769356
Research Article
The Immune Response of Acanthaster planci to Oxbile Injections and Antibiotic Treatment


Am J Cancer Res 2013;3(3):278-289
www.ajcr.us /ISSN:2156-6976/ajcr0000201
The C-terminal common to group 3 POTES (CtG3P): a 
newly discovered nucleolar marker associated with 
malignant progression and metastasis

Nature America Inc. • http://biotech.nature.co
Molecular breeding of carotenoidbiosynthetic pathways

J Biol Chem. Apr 11, 2014; 289(15): 10930–10938.
Published online Feb 25, 2014. doi: 10.1074/jbc.M113.533216
PMCID: PMC4036204
The DDN Catalytic Motif Is Required for Metnase Functions in Non-homologous End Joining (NHEJ) Repair and Replication Restart

Transl Psychiatry. Nov 2013; 3(11): e323.
Published online Nov 5, 2013. doi: 10.1038/tp.2013.96
PMCID: PMC3849963
Derivation of neural stem cells from an animal model of psychiatric disease

Experimental and Molecular Pathology
Available online 30 July 2014
In Press
Systemic distribution, subcellular localization and differential expression of sphingosine-1-phosphate receptors in benign and malignant human tissues

Journal of Advanced Research
Available online 22 May 2014
In Press
ORIGINAL ARTICLE
Expression, genetic localization and phylogenic analysis of NAPlr in piscine Streptococcus dysgalactiae subspecies dysgalactiae isolates and their patterns of adherence

ORIGINAL RESEARCH ARTICLE Front. Immunol., 17 September 2013 | doi: 10.3389/fimmu.2013.00287
Th1/M1 conversion to Th2/M2 responses in models of inflammation lacking cell death stimulates maturation of monocyte precursors to fibroblasts

Hindawi Publishing Corporation
Journal of Marine Biology
Volume 2014, Article ID 769356, 11 pages
http://dx.doi.org/10.1155/2014/769356
Research Article
The Immune Response of Acanthaster planci to
Oxbile Injections and Antibiotic Treatment

McMaster University © by Dipannita Basu, July 2013
CHARACTERIZATION OF THE MECHANISM OF ACTION FOR NOVEL 
ALLOSTERIC MODULATORS OF THE DOPAMINE D2 RECEPTOR

J CARDIOVASC PHARMACOL THER 2013 18: 280 originally published online 14 December 2012
Probenecid as a Noninjurious Positive Inotrope in an Ischemic Heart Disease Murine Model

Stem Cell Reviews and Reports
August 2014
Date: 05 Aug 2014
Motor Neuron Differentiation from Pluripotent Stem Cells and Other Intermediate Proliferative Precursors that can be Discriminated by Lineage Specific Reporters poly-d-lysine coating coverslips


The Royal Society of Chemistry
2015,
Research Article
Cationic Lipsomes as efficient nanocarriers for the drug delivery of an anticancer cholesterol-based ruthenium complex


Carroll College Natural Sciences Department
Helena, Montana
Exploring the Role of Olfm1 in the Trafficking of GluR2- containing AMPA Receptors

Published: August 06, 2013 DOI: 10.1371/journal.pone.0070736
Effects of the Dopamine D2 Allosteric Modulator, PAOPA, on the Expression of GRK2, Arrestin-3, ERK1/2, and on Receptor Internalization

PLOS ONE | www.plosone.org
Dynein Light Chain 1 (DYNLT1) interacts with
normal and oncogenic nucleoporins

Am J Cancer Res. 2013; 3(3): 278–289.
Published online Jun 20, 2013.
PMCID: PMC3696534
The C-terminal common to group 3 POTES (CtG3P): a newly discovered nucleolar marker associated with malignant progression and metastasis

Cell Rep. May 22, 2014; 7(4): 982–988.
Published online May 15, 2014. doi: 10.1016/j.celrep.2014.04.020
Calcineurin is universally involved in vesicle endocytosis at neuronal and non-neuronal secretory cells polylysine coating

J Neurosci. May 22, 2013; 33(21): 9169–9175.
doi: 10.1523/JNEUROSCI.0301-13.2013
The SNARE proteins SNAP25 and synaptobrevin are involved in endocytosis at hippocampal synapses

Nature Communications 5, Article number: 3356 doi:10.1038/ncomms4356
Post-fusion structural changes and their roles in exocytosis and endocytosis of dense-core vesicles

Nat Commun. 2014 Jun 17;5:4112. doi: 10.1038/ncomms5112.
Senescence impairs direct conversion of human somatic cells to neurons.
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Nat Neurosci. 2014 Feb;17(2):232-9. doi: 10.1038/nn.3615. Epub 2014 Jan 5.
Presynaptic glycine receptors as a potential therapeutic target for hyperekplexia disease.

J Natl Compr Canc Netw 2014;12:889-897
Optimal First-Line and Maintenance Treatments for Advanced-Stage Nonsquamous Non-Small Cell Lung Cancer

Semin Oncol. 2014 Feb;41(1):93-100. doi: 10.1053/j.seminoncol.2013.12.007. Epub 2013 Dec 12.
Chemotherapy and targeted therapeutics as maintenance of response in advanced non-small cell lung cancer.

Ther Adv Med Oncol. 2014 Jan;6(1):4-15. doi: 10.1177/1758834013510589.
Maintenance treatment after induction therapy in non-small cell lung cancer: latest evidence and clinical implications.

Curr Treat Options Oncol. 2013 Dec;14(4):595-609. doi: 10.1007/s11864-013-0255-3.
Bevacizumab in advanced NSCLC: chemotherapy partners and duration of use

Cancer Res. 2014 Jun 13.
Germline Mutation of Bap1 Accelerates Development of Asbestos-Induced Malignant Mesothelioma.

J Cardiovasc Pharmacol. 2013 Apr;61(4):311-7. doi: 10.1097/FJC.0b013e318280e16e.
Sulfur-containing angiotensin-converting enzyme inhibitor 3-thienylalanine-ornithyl-proline activates endothelial function and expression of genes involved in Renin-Angiotensin system.

Cancer Res. 2014 Mar 1;74(5):1390-403. doi: 10.1158/0008-5472.CAN-13-1275.
LIMD2 is a small LIM-only protein overexpressed in metastatic lesions that regulates cell motility and tumor progression by directly binding to and activating the integrin-linked kinase.

Cell Tissue Res. 2014 May 28.
MicroRNA-dependent genetic networks during neural development.


Oxford Journals Volume 23 Issue I, P145-156

Research Article
Hexokinase activity is required for recruitment of parkin to depolarized mitochondria


Cancer Research, June 1, 2014
Fatty acid-binding protein E-FABP restrcts tumor growth by promoting IFN-b responses in tumor-associated macrophages

1.5H high performance coverslips

 

Molecular Reproduction and Development

Volume 81, Issue 12

Abstract
Kpna7 interacts with egg-specific nuclear factors in the rainbow trout (oncorhynchus mykiss)

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Protocol for primary neuron cultures

Day 1:

Add poly D-lysine/laminin solution to 24-well plate. The solution contains poly D-lysine at a final concentration of 0.05 mg/ml and laminin at a final concentration of 0.005 mg/ml. Swirl the plate to ensure that the coating mix covers the entire bottom of the plate. Leave the dishes/coverslips in the 37oC/5% CO2 incubator overnight.

Day 2:

1.Wash the dishes/coverslips twice with sterile water; remove the final wash and leave them liquid-free in the incubator.

2.Make up Neuronal Growth Media with serum:•911 mL BME Eagle media without L glutamine and with Earle salts•50 mL bovine calf serum•24 mL 1_1/2 solution (To 97.6 mL of H2O, add 1.4 mL of 2.5 M glucose solution, 0.5 mL of 0.2 M L-glutamine solution, and 0.5 mL of Pen/Strep solution)•4.5 mL Stable Vitamin Mix (To 198 mL of distilled water, mix in the following by stirring: 600 mg L-proline (Sigma P-0380), 600 mg L-cysteine (Sigma C-8755), 200 mg p-aminobenzoic acid (Sigma A-9879), 80 mg vitamin B12 (Sigma V-2876), 400 mg i-inositol (meso)(Sigma I-5125), 400 mg choline chloride (Sigma C-1879), 1 g fumaric acid (Sigma F-2752), and 16 mg coenzyme A (Pharmacia 28-3001-02). Then add the following to 10 mL of distilled water: 0.4 mg d-biotin (Sigma B-4501) and 100 mg DL-6,8-thioactic acid (Sigma T-5625). Shake to resuspend. Then quickly pipette out 2 mL of the biotin solution and add it to the 198 mL solution above. The ingredients do not dissolve completely, so stir before aliquoting into Neuronal Growth Media). •0.5 mL ITS (stock solution is 5 mL H2O added to Sigma product I1884)•5 mL Putrescine (1.6 mg/mL stock solution using Sigma product P7505)•5 mL Transferrin (5 mg/mL stock solution using Sigma product T2252)•100 uL Progesterone (1.2 mM stock solution using Sigma product P6149).

3.Make up Optimem/glucose solution (add 4 mL of 2.5 M glucose to 500 mL Optimem (Invitrogen)).4.Make up DM/KY, sterile filter, and place on ice. Make the DM/KY slightly basic (just pinkish, approximate pH of 7.5-7.6). During the dissection process, you’ll notice that the DM/KY solution next to brains in the dish turns yellow. This is from lactic acid release. Making the DM/KY slightly more basic helps better neutralize this massive lactic acid release.Composition of DM/KY solution:Dilute the 10x KY stock prepared below into the appropriate volume of DM. To make 1000ml DM, add the ingredients below to distilled water for a total volume of 1000 ml and then filter sterilize. Store at 4oC in refrigerator.DM IngredientFinal concentrationStock concentrationVolume toadd for 1000 mLNa2SO481.8 mM1 M81.8 mLK2SO430 mM0.5 M60 mLMgCl25.8 mM1 M5.8 mLCaCl20.25 mM0.1 M2.52 mLHEPEs1 mM1 M1 mLGlucose20 mM2.5 M8 mLPhenol Red0.001%0.5%2 mLNaOH0.16 mM0.1 N1.6 MlFor 10x KY solution, gradually add small amounts of kynurenic acid to water containing phenol red and use the color of the phenol red to titrate the pH of the solution back up to about 7.4 as the acid dissolves. Filter the mixture of ingredients below and store at 4oC.10X KY IngredientFinal concentrationStock concentrationVolume to add for 1000 mLKynurenic acid10 mM1.8925 gPhenol Red0.0025%0.5%5 mLHEPES5 mM1 M5 mLMgCl2100 mM1 M100 mLNaOH1 NAdd dropwise to titrate pH5.Make up the trypsin inhibitor solution and the papain solution BUT DO NOT add papain at this point; place solutions on ice. To prepare the solutions, add 150 mg of trypsin inhibitor to 10 mL of DM/KY and pH the solution until it is again slightly basic (estimate a pH of 7.5-7.6). Add 2-3 mg of cysteine to 10 mL of DM/KY to make the papain solution and again pH to around 7.5-7.6. Leave these solutions at room temperature.6.Pour ice-cold DM/KY solution into several culture dishes: 1 large dish for the pups and 10cm dishes for the pup heads, for the intact brains and for the dissected striatum. Place dishes on ice. Pour all solutions under the hood to keep things as sterile as possible for as long as possible.

7.Put all the dissection tools you will be working with (several pairs of forceps, a chemical spatula, one large and one small pair of scissors, and anything else you will need) into an alcohol bath to sterilize. If infections have been a problem, consider flame sterilizing the dissection tools and leaving them on the edge of a surface so that the parts that touch the rat embryos don’t touch any unsterilized surface. While you are doing your dissections below, be sure to place your dissection tools (when not in use) in a way that maintains their relative sterility. One way to do this is to have the top cover of a 15 cm dish and place the forceps/scissors/spatula on the cover such that the end of the instruments that will be touching the rat brains is hanging off the edge of the dish cover.

8.Obtain pregnant rat Dissection of striatum:

1.Sacrifice the rat.

2.After the rat fails to move spontaneously or in response to pain (touch the eye and look for a reflex), puncture each lung with a needle. Clean the belly of the rat with alcohol and then incise along the abdomen and remove the uterus. Place the pups into the large culture dish on ice. Try hard to prevent the pups from touching the outside of the mother or other unsterile surfaces. The better you can get the pups straight into the large culture dish, the better.

3.Remove the heads of the pups and place in a 10cm dish on ice.

4.The rest of the protocol is done on ice, but under a dissection microscope. For each head, remove the skin and place the two prongs of one set of forceps into and through the eyes of the head (the head should be positioned so you are looking down on the top of the head). This pins the head down and allows you to use the other set of forceps to cut into the head. Use the other forceps (in a closed position) to puncture the part of the skull that is exactly midline where all the sutures meet. Then carefully run your forceps towards the eyes and then towards the back of the head to open up a midline cut in the skull (don’t dig down too far or you’ll scrape brain). Once you have a big enough opening, remove your other pair of forceps from their position through the eyes and peel the skull back using each forceps, pulling in opposite directions; this kind of counter-traction is most effective in breaking open the skull. Once enough of the brain is exposed, take the chemical spatula, dig underneath the brain, and scoop the brain out. Place the brain into a new 10 cm dish with DM/KY on ice.5.Repeat the process, but BE SURE that the dish which contains your newly dissected brains gets swirled every once in a while. This prevents the local buildup of lactic acid around each brain, which decreases viability of your neurons.6.Once you have removed all the brains, you are ready to dissect the striatum. Orient yourself so that the brain is facing forward (the olfactory bulbs are at the top of your view and you are looking down at the top of the brain, rather than looking down on the brainstem). For each hemisphere, use your forceps to dissect longitudinally (sagittal incision) down the hemisphere. There should be equal amount of cortex to the left and to the right of your cut. Don’t dissect too deep. Your goal is to just expose the structures underneath the superficial cortex. When you’ve split open the surface of the cortex, you should eventually be able to see the fine capillary network that makes up the choroid plexus of the lateral ventricles (you may need to very gently use the blunt aspect of both forceps to open up the longitudinal cut you’ve made to see the plexus. Once you’ve located the plexus, you want to push your forceps down into the incision you made such that you are splitting the cortices exactly along the plane where the choroid plexus is. This will leave you with a lateral half of the cortical hemisphere. Once you have isolated this lateral half of the cortical hemisphere, place it so that the side facing you was previously buried in the brain and the side facing the dish is the lateral surface of the cortex. From there, you should be able to faintly see a semi-circle like structure. The cortex will appear to slightly indent (like it has folded over something else) on the side closest to the top of the brain (i.e. not the side that was previously buried). We will call this side the lateral surface. The indentation of the cortex near the lateral surfacecreates a semi-circle structure. The striatum is the brain material that is medial to this semi-circle indentation. You may see the hippocampus posteriorly (it looks like a banana – a curvy line inside a banana-peel). Be sure to dissect away the hippocampus. Cut along the semi-circle line and throw out the cortex and hippocampus that came off. You should now be left with a semi-circle piece of tissue that is MAINLY striatum. However, the cortex actually coats the piece of tissue you have ON THE BACK SIDE (i.e. the side facing the dish). You need to be sure to dissect this remaining cortex off the striatum. Do this by placing the semi-circle piece of tissue such that the medial aspect of the semi-circle (the part of the semi-circle that was oriented towards the bottom of the culture dish when the brain was intact) is again facing down towards the bottom of the dish. This now means that the lateral edge of the semi-circle is on top and closest to your eyes. You should be able to discern a faint tissue plane in looking at the tissue from this angle. The lateral side of that tissue plane is the cortex that is wrapped around the back of the striatum. Use your forceps to dissect down through this tissue plane to remove the cortex that has coated the striatum. Take your piece of freshly dissected striatum and place it in a new 10 cm dish with DM/KY on ice.

7.Again, every once in a while, be sure to swirl both the dish that has the brains in it and the dish that has just the striatum in it to help prevent local buildup of lactic acid.

8.Repeat the process on the other cortical hemisphere. When you have just 1-2 brains left to dissect, take a break and add papain to the papain solution. You should be adding 100 units of papain to the solution. Place both the papain solution and the trypsin inhibitor solution in the 37oC water bath. Be sure the water bath is actually at 37 degrees!

9.Finish your dissections.

10.Sterile filter the papain and trypsin inhibitor solutions. Leave both solutions out at room temperature.

11.Transfer the striatal tissue to a 15ml conical tube taking as little DM/KY as possible. Once the tissue has settled remove the extra DM/KY solution. Papain treatment:1.Add 10ml of the papain solution to the dissected tissue and incubate at 37oC for 15min, mixing every 5min. 2.Remove the enzyme solution.Trypsin Inhibitor treatment:

1.Add 5 ml of trypsin inhibitor, mix the tissue, and incubate for 10 min at ROOM TEMPERATURE.

2.After 10 min, remove the trypsin inhibitor and replace with a fresh 5 ml aliquot. Wait another 10 min at room temperature.

3.Remove trypsin inhibitor solution and wash with 10 mL of Optimem / glucose; the Optimem/glucose should be at room temperature. Remove the Optimem/glucose solution.

Trituration:

1.Add 5 mL of Optimem/glucose. Triturate gently with a 5 ml pipette until the solution turns cloudy. The trituration should start off extremely slow. You have to be patient during these steps of the protocol since your yield and neuronal health will go up substantially if you take your time and triturate slowly. Also, during your first few rounds of trituration, you should avoid pipetting the brain/Optimem solution directly into the bottom of the conical tube. Instead, as you pipette out, you should be simultaneously lifting your pipette higher and higher out of the conical tube. The tip of the pipette should still stay under the Optimem solution, but it shouldn’t be too far under the surface of the solution. Triturate until the solution becomes somewhat cloudy (but not too cloudy!). If you triturate up and down with dissociated neurons (which make the solution cloudy), you’ll cause sheering stress and kill them. So the goal is to free up enough neurons each round, but stop the trituration as soon as there are a reasonable number of neurons free in solution.

2.Allow the brain material to settle (at room temperature) and then take the cloudy supernatant and transfer it to a 50 mL conical tube that is also left at room temperature. Add 5 mL of new Optimem/glucose to the 15 mL conical tube and repeat trituration.

3.Keep repeating steps 1 and 2. As you increase the number of times you triturate, you will eventually have to triturate more aggressively (faster and pipetting against the wall of the conical tube). You should only become more aggressive when gentler methods fail to turn the solution cloudy and you still have significant brain material left.

4.I typically triturate 10 times for striatum collected from 10-15 brains.

5.Allow everything to settle in the 50 mL conical tube and then take a pipette and suck up the random debris/DNA/etc. that has accumulated at the bottom of the tube. Be careful not to contaminate the solution with any unsterile parts of the pipette (e.g. if you are using a P1000 to suck up the left-over bits in the 50 mL conical tube, the sterile tip may not reach all the way to the bottom of the conical tube).

Plating cells:

1.Mix the cell suspension in the 50 mL conical tube and transfer a 10ul aliquot to a tube that contains 10 ul of DM/KY and 10 ul of Trypan blue. Mix thoroughly, add to the cytometer, and count the number of cells in the 16 box squares in the 2 opposite corners of the field. Average the 2 counts or recount if the 2 numbers are different by more than 10%. Multiply the average by 30, 000 to get the number of cells per ml.

2.Dilute the cells with Optimem/glucose solution to a final count of 0.5-0.6 million per mL and plate 1 mL per 24 well plate.

3.As you are plating, be sure to add the neurons to the center of the well (don’t squirt the cell suspension solution onto the side of the well). Also, after adding the cell suspension solution to all the wells in a row, swirl the plate to make sure the neurons are evenly distributed and swirl your dispensing container to make sure the neurons don’t settle.

4.Once you have fully plated the neurons, swirl the plate one last time and then don’t move the plate at all. Just leave the plate in the hood (at room temperature) for about an hour. This helps ensure that the neurons settle evenly across the whole well. We’ve previously had problems with the neurons settling preferentially in the center of the well or the periphery of the well.

5.After an hour, place the plates in the 37 degree incubator and leave for another hour.

6.Check the plates under the microscope to ensure that the neurons have adhered to the well surface (tap the plate and observe for movements of the neurons under the scope).

7.Assuming the neurons have attached, replace the Optimem/glucose with pre-warmed Neuronal Growth Media (that includes serum).