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1、 ScienCell Research LaboratoriesTMHuman Brain Microvascular Endothelial Cells (HBMEC) Catalog Number: 1000 Cell Specification The brain microvascular endothelial cells (MEC) are the major element of the blood-brain barrier and comprise the primary limitation to passage of substances, both soluble an
2、d cellular, from the blood into the brain. Brain MEC utilizes unique features that distinguish themselves from those of peripheral endothelial cells. Most prominent among these are the following: 1) intercellular tight junctions that display high transendothelial electrical resistance and retard par
3、acellular flux 1, 2) absence of fenestrae and a reduced level of fluid-phase endocytosis 2, and 3) asymmetrically-localized enzymes and carrier-mediated transport systems that engender a truly polarized phenotype 3. Like peripheral endothelial cells, however, brain MEC express, or can be induced to
4、express, cell adhesion molecules on their surface that regulate the extravasation of leukocytes into the brain. In view of the organ specificity of MEC, Brain MEC has been widely used for studying the molecular and cellular basis of blood-brain barrier. HBMEC from ScienCell Research Laboratories are
5、 isolated from human brain tissue. HBMEC are cryopreserved at passage one and delivered frozen. Each vial contains 5 x 105 cells in 1 ml volume. HBMEC are characterized by immunofluorescent method with antibodies to vWF/Factor VIII and CD31 (P-CAM) and by uptake of DiI-Ac-LDL. HBMEC are negative for
6、 HIV-1, HBV, HCV, mycoplasma, bacteria, yeast and fungi. HBMEC are guaranteed to further culture in the conditions provided by ScienCell Research Laboratories. Recommended Medium It is recommended to use Endothelial Cell Medium (ECM, Cat. No. 1001) for the culturing of HBMEC in vitro. Product Use HB
7、MEC are for research use only. It is not approved for human or animal use, or for application in in vitro diagnostic procedures. Storage Directly and immediately transfer cells from dry ice to liquid nitrogen upon receiving and keep the cells in liquid nitrogen until cell culture needed for experime
8、nts. Shipping Dry ice. Reference 1 Crone, C. and Oleson, S. P. (1992) Electrical resistance of brain microvessel endothelium. Brain Res. 241: 49- 55. 2 Reese, T. S. and Karnovsky, M. J. (1967) Fine structural localization of blood-brain barrier to exogenous peroxidase. J. Cell Biol. 34:9-14. 3 Vorbr
9、odt, A. W. (1988) Ultrastructural cytochemistry of blood-brain barrier endothelia. Prog. Histochem. Cytochem. 18(3):1-96. Instruction for culturing cells Caution: Cryopreserved cells are very delicate. Thaw the vial in a 37oC waterbath and return them to culture as quickly as possible with minimal h
10、andling! Set up culture after receiving the order: 1. Prepare a fibronectin coated flask (2 g/cm2, T-75 flask is recommended). Add 10 ml of sterile Dulbeccos phosphate buffered saline (DPBS) to a T-75 flask and then add 150 l of fibronectin stock solution (1 mg/ml, Sigma cat. no. F1141). Leave the f
11、lask in incubator overnight. 2. Prepare complete medium: decontaminate the external surfaces of medium and medium supplements with 70% ethanol and transfer them to sterile field. Aseptically open each supplement tube and add them to the basal medium with a pipette. Rinse each tube with medium to rec
12、over the entire volume. 3. Aspirate fibronectin solution and add 20 ml of complete medium to the flask. Leave the flask in the hood and go to thaw the cells. The fibronectin solution can be used twice. 4. Place the vial in a 37oC waterbath, hold and rotate the vial gently until the contents are comp
13、letely thawed. Remove the vial from the waterbath immediately, wipe it dry, rinse the vial with 70% ethanol and transfer it to a sterile field. Remove the cap, being careful not to touch the interior threads with fingers. Using a 1 ml eppendorf pipette gently resuspend the contents of the vial. 5. D
14、ispense the contents of the vial into the equilibrated, fibronectin-coated flask. A seeding density of 7,500 cells/cm2 is recommended. Note: Dilution and centrifugation of cells after thawing are not recommended since these actions are more harmful to the cells than the effect of DMSO residue in the
15、 culture. It is also important that endothelial cells are plated in fibronectin coated flask that promotes cell attachment. 6. Replace the cap or cover of flask, and gently rock the flask to distribute the cells evenly. Loosen cap if necessary to permit gas exchange. 7. Return the culture vessels to
16、 the incubator. 8. For best results, do not disturb the culture for at least 16 hours after the culture has been initiated. Change the growth medium the next day to remove the residual DMSO and unattached cells, then every other day thereafter. A healthy culture will display cobblestone or spindle shaped morphology, nongranular cytoplasm and the cell number will be double after two to three days in culture. Maint
