Immortalized Mouse Cerebral Capillary Endothelial Cells (cEND)

CAT.NOUNITPRICE
T02901x106 cells / 1.0 ml
$0.00

Specifications


Description

Homeostasis of the central nervous system (CNS) is maintained by the blood brain barrier (BBB) and disruptions to the BBB are linked to many disorders of the CNS. The Immortalized Mouse Cerebral Capillary Endothelial Cell Line (cEND) provides a useful model for studies involving the differentiation and regulation of BBB as it shares principal features of the BBB in vivo, specifically in that 1) it retains the endothelial markers VE-Cadherin and PECAM-1; 2) it displays tight junction associated markers such as occludin, claudin-3, -5, -12, 3) it expresses the BBB marker protein Glut-1 and 4) it shows high electrical resistance, representing barrier properties. This cell line is also responsive to glucocorticoid, estrogen-treatment and pro-inflammatory mediator such as TNFα, making this cell line valuable in elucidating cellular responses of endothelial cells to different stimuli. Together with the Immortalized Mouse Cerebellar Capillary Endothelial Cell Line (cerebEND), the two cell lines represent important in vitro model system for these different brain regions.

SKUT0290
SpeciesMouse (M. musculus)
Species descriptionNeonatal mouse (WT 129 Sv strain)
Tissue/Organ/Organ SystemBrain
Growth PropertiesAdherent
Cell MorphologySpindle shaped
Seeding Density30,000 - 35,000 cells/cm2; Recommended split ratio: no greater than 1:4
Population Doubling Time60 - 80 hours
Immortalization MethodTransformation with oncoprotein of murine Polyomavirus, Polyoma middle T antigen (PymT)
Applications

For Research Use Only

Unit quantity1x106 cells / 1.0 ml
Cell TypeImmortalized Cells
Expression Profile

Claudin-5, Occuldin, Glut-1, VE-Cadherin

Propagation Requirements
Use of PriCoatTMT25 Flasks (G299) or Applied Cell Extracellular Matrix (G422) is required for cell adhesion to the culture vessels. Grow cells inECM-coated culture vessels unless otherwise specified in the Propagation Requirements below.

The base medium for this cell line is Prigrow III medium available at abm, Cat. No. TM003. To make the complete growth medium, add the following components to the base medium: heat-inactivated fetal bovine serum to a final concentration of 10% and Penicillin/Streptomycin Solution (G255) to a final concentration of 1%.
Change media every 2-3 days.
Carbon dioxide (CO2): 5%, Temperature: 37.0°C.

The Differentiation Medium is composed of 2% heat-inactivated fetal bovine serum instead of 10%. Culturing cEND cells in serum-reduced medium leads to an increase in TER (from 150 Ωcm2 in the presence of 10% serum to 500 Ωcm2 in the presence of 2% serum), as well as change in cell morphology (from spindle-shaped to cobble-stone like). TER can be further increased by the addition of 110nM hydrocortisone (800 Ωcm2 ) or 1µM insulin (1000 Ωcm2 ) into the differentiation medium.

QC

1) Endothelial marker VE-Cadherin and PECAM-1 assessed by immunostainning; 2) Tight junction protein Claudin-5 assessed by immunostainning and claudin-1, -3 and -12 detected in mRNA level; 2) BBB marker protein Glut-1 and tight junction-associated protein occludin measured by western blot analysis. The cell line's response to pro-inflammatory stimuli was measured by 1) TER, 2) western blot and 3) gene expression analysis using RT-PCR.

Disclaimer

1. For for-profit organizations and corporations, please contact [email protected] for pricing of this item.

2. Sale of this item is subjected to the completion of a Material Transfer Agreement (MTA) by the purchasing individual/institution for each order. If you have any questions regarding this, please contact us at [email protected].

3. All test parameters provided in the CoA are conducted using abm's standardized culture system and procedures. The stated values may vary under the end-user's culture conditions. Please verify that the product is suitable for your studies by referencing published papers or ordering RNA (0.5 μg, Cat.# C207, $450.00) or cell lysate (100 μg, Cat.# C206, $600.00) to perform preliminary experiments, or alternatively use our Gene Expression Assay Service (Cat# C138). All sales are final.

4. We recommend live cell shipments for ease of cell transfer and this option can be requested at the time of ordering. Please note that the end-user will need to evaluate the feasibility of live cell shipment by taking into account the final destination's temperature variation and its geographical location. In addition, we thoroughly test our cell lines for freeze-thaw recovery. If frozen cells were received and not recovered in your lab under the exact, specified conditions (using recommended culture vessel, media, additional supplements, and atmospheric conditions), a live cell replacement is possible at a cost (plus shipping).

5. All of abm's cell biology products are for research use ONLY and NOT for therapeutic/diagnostic applications. abm is not liable for any repercussions arising from the use of its cell biology product(s) in therapeutic/diagnostic application(s). Please contact a technical service representative for more information.

6. abm makes no warranties or representations as to the accuracy of the information on this site. Citations from literature and provided for informational purposes only. abm does not warrant that such information has been shown to be accurate.

7. abm warrants that cell lines shall be viable upon initiation of culture for a period of thirty (30) days after shipment and that they shall meet the specifications on the applicable abm Material Product Information sheet, certificate of analysis, and/or catalog description. Such thirty (30) day period is referred to herein as the "Warranty Period."

DepositorUniversity of Würzburg
FAQs


I want to make sure these cells express my gene of interest before I decide to buy the cell line. Can you provide a sample so this can be tested?
We do not carry out downstream characterization or gene expression profiling of our cell lines. To facilitate your preliminary experiments we can provide an RNA extraction (0.5ug total RNA) or cell lysate (100ug/100ul provided in 62.5mM Tris‐HCl, 2% SDS, 10% Glycerol, 50mM DTT, 0.01% w/v Bromophenol Blue) for any of our immortalized cell lines for a small fee. Please inquire directly for more information. The lead time will be around 2 weeks from the time of placing an order (if the item is in stock).
How often do I need to change the media?
The media should be changed every 2-3 days.
Why do these cells need bio safety level II?
In order to be more cautious, we follow the CDC-NIH recommendations that all mammalian sourced products should be handled at the Biological Safety Level 2 to minimize exposure of potentially infectious products. This information can be found in 'Biosafety in Microbiological and Biomedical Laboratories' (1999). Your institution's Safety Officer or Technical Services will be able to make the call as to whether BioSafety Level I is possible with these cells at your site if required.
Do you sell ECM coated T75 flasks?
Yes we can provide a coating service. Please inquire with [email protected]
What can I coat a larger dish to subculture?
We also offer applied extracellular matrix (collagen type I) in liquid form, for the coating of larger flasks and other required plasticware: http://www.abmgood.com/Applied-Cell-Extracellular-Matrix-G422.html
How long can I store frozen vials for?
Cells that are properly frozen using an effective cryoprotective agent can be stored in liquid nitrogen indefinitely without affecting their recovery.
Should the cap of the flask be changed before starting the cell culturing step?
No, there is no need in sterile biosafety cabinets unless it has contacted any non-sterile condition (e.g. touching the contaminated tip, etc.).
What is the recommended storage temperature?
In general, if you received: Live cells: acclimatize for 3-4 hrs at at the recommended conditions stated for the cell line under the propagation section, and then change media afterwards. Frozen cells: Immediately place cells in liquid nitrogen; -180C.
How is cell density crucial for drug selection?
If antibiotic selection is applicable to the target cells, we suggest getting rid of all the background cells so that the cell density is kept lower (even 20-30%). However, once the clones are selected by clonal dilution, we don't need the drug to still be present. If needed, the cell density should be towards the higher end since cells are already selected. Any primary cells still present will be depleted as a result of senescence and the cell population that remains will be resistant to the specific antibiotic.
My cells are not detaching, what method do you recommend to trypsinize the cells?
1. Incubate the coated plate containing trypsin solution at recommended temperature indicated in the propagation section for 3-5 min till the cells round up, monitoring from time to time under microscope. 2. Diluting G422 (1:1) with PBS and coating for lesser time. Sometimes the collagen content in G422 is higher and thus make stronger bonding with cells. 3. You can try reducing the incubation time as well for coating the plate to make a thinner layer.
Why is it important to determine the optimal seeding density?
The seeding density we recommend is for when cells are plated to a new vessel. The optimal seeding density should allow cells to attach to the surface and have room to proliferate. If you seed too little, cells may not attach well to the surface (for adherent cells). Seeding density is important as many cells (adherent or suspension cells) need to be in close proximity for better growth. Cell-cell interactions allow cells to communicate with each other in response to changes in their microenvironment. This ability to send and receive signals is essential for the survival of the cell. In other cases, if the seeding density is too low, cells may attach but a retardation in cell growth is observed. If you seed too high, the cells will attach but there is insufficient room for further proliferation and they will stop replicating.
References


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  • Salvador, E et al. "Stretch in brain microvascular endothelial cells (cEND) as an in vitro traumatic brain injury model of the blood brain barrier" J Vis Exp 80:e50928 (2013). DOI: 10.3791/50928. PubMed: 24193450.
  • Burek, M et al. "Generation of an immortalized murine brain microvascular endothelial cell line as an in vitro blood brain barrier model" J Vis Exp 66:e4022 (2012). DOI: 10.3791/4022. PubMed: 22951995.
  • Dakwar, GR et al. "Toxicity assessment of extracts from infusion sets in cEND brain endothelial cells" Int J Pharm 434(1-2):20-7 (2012). DOI: 10.1016/j.ijpharm.2012.05.019. PubMed: 22643027.
  • Blecharz, KG et al. "Glucocorticoid effects on endothelial barrier function in the murine brain endothelial cell line cEND incubated with sera from patients with multiple sclerosis" Mult Scler 16(3):293-302 (2010). DOI: 10.1177/1352458509358189. PubMed: 20203147.
  • Burek, M et al. "Cloning and characterization of the murine claudin-5 promoter" Mol Cell Endocrinol 298(1-2):19-24 (2009). DOI: 10.1016/j.mce.2008.09.041. PubMed: 18996436.
  • Prinz, M et al. "1,4-Substituted 4-(1H)-pyridylene-hydrazone-type inhibitors of AChE, BuChE, and amyloid-β aggregation crossing the blood-brain barrier" Eur J Pharm Sci 49(4):603-13 (2013). DOI: 10.1016/j.ejps.2013.04.024. PubMed: 23643737.
  • Harke, N et al. "Glucocorticoids regulate the human occludin gene through a single imperfect palindromic glucocorticoid response element" Mol Cell Endocrinol 295(1-2):39-47 (2008). DOI: 10.1016/j.mce.2008.08.011. PubMed: 18782596.
  • Blecharz, KG et al. "Glucocorticoids increase VE-cadherin expression and cause cytoskeletal rearrangements in murine brain endothelial cEND cells" J Cereb Blood Flow Metab 28(6):1139-49 (2008). DOI: 10.1038/jcbfm.2008.2. PubMed: 18231113.
  • Förster, C et al. "Dexamethasone induces the expression of metalloproteinase inhibitor TIMP-1 in the murine cerebral vascular endothelial cell line cEND" J Physiol 580(Pt.3):937-49 (2007). PubMed: 17317742.
  • Silwedel, C et al. "Differential susceptibility of cerebral and cerebellar murine brain microvascular endothelial cells to loss of barrier properties in response to inflammatory stimuli" J Neuroimmunol 179(1-2):37-45 (2006). PubMed: 16884785.
  • Förster, C et al. "Occludin as direct target for glucocorticoid-induced improvement of blood-brain barrier properties in a murine in vitro system" J Physiol 565(Pt 2):475-86 (2005). PubMed: 1579066.
  • Förster, C et al. "Glucocorticoid effects on mouse microvascular endothelial barrier permeability are brain specific" J Physiol 573(Pt 2):413-25 (2006). PubMed: 16543270.
  • Kleinschnitz, C et al. "Glucocorticoid insensitivity at the hypoxic blood-brain barrier can be reversed by inhibition of the proteasome" Stroke 42(4):1081-9 (2011). DOI: 10.1161/STROKEAHA.110.592238. PubMed: 21330632.
  • Salvador, E et al. "Glucocorticoids and endothelial cell barrier function" Cell Tissue Res 355(3):598-605 (2014). DOI: 10.1007/s00441-013-1762-z. PubMed: 24352805.
  • Burek, M et al. "Claudin-5 as a novel estrogen target in vascular endothelium" Arterioscler Thromb Vasc Biol 30(2):298-304 (2010). DOI: 10.1161/ATVBAHA.109.197582. PubMed: 19910637.
  • Burek, M et al. "Mechanisms of transcriptional activation of the mouse claudin-5 promoter by estrogen receptor alpha and beta" Mol Cell Endocrinol 392(1-2):144-51 (2014). DOI: 10.1016/j.mce.2014.05.003. PubMed: 24846172.
  • Förster, C et al. "Tight junctions and the modulation of barrier function in disease" Histochem Cell Biol 130(1):55-70 (2008). DOI: 10.1007/s00418-008-0424-9.