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«GUIDELINES FOR USE PRODUCT: Corning® Matrigel® Basement Membrane Matrix, 5 mL vial CATALOG NUMBER: 356234 BACKGROUND: Basement membranes are thin ...»

GUIDELINES FOR USE

PRODUCT: Corning® Matrigel® Basement Membrane Matrix, 5 mL vial

CATALOG NUMBER: 356234

BACKGROUND: Basement membranes are thin extracellular matrices underlying cells in vivo. Corning

Matrigel Basement Membrane Matrix is a solubilized basement membrane preparation

extracted from the Engelbreth-Holm-Swarm (EHS) mouse sarcoma, a tumor rich in

extracellular matrix proteins. Its major component is laminin, followed by collagen IV, heparan sulfate proteoglycans, entactin/nidogen.1,2 Corning Matrigel Basement Membrane Matrix also contains TGF-beta, epidermal growth factor, insulin-like growth factor, fibroblast growth factor, tissue plasminogen activator, 3,4 and other growth factors which occur naturally in the EHS tumor. Corning Matrigel Basement Membrane Matrix is effective for the attachment and differentiation of both normal and transformed anchorage dependent epithelioid and other cell types. These include neurons, 5,6 hepatocytes,7 Sertoli cells,8,9 chick lens,10 and vascular endothelial cells.11 Corning Matrigel Basement Membrane Matrix will influence gene expression in adult rat hepatocytes, 12,13 vascular endothelial cells,14 as well as three dimensional culture in mouse15-18 and human19,20 mammary epithelial cells. It is the basis for several types of tumor cell invasion assays,21,22 will support in vivo peripheral nerve regeneration, 23-25 and provides the substrate necessary for the study of angiogenesis both in vitro26,27 and in vivo.25, 28-30 Corning Matrigel Basement Membrane Matrix also supports in vivo propagation of human tumors in immunosupressed mice.31-33 Corning Matrigel Basement Membrane Matrix can be used for the transplantation of unsorted mammary cells, 34 as well as sorted epithelial subpopulations embedded in Corning Matrigel.35,36 This matrix has also been used as a cancer stem cell model and shown to enhance tumor growth rates in vivo.37 SOURCE: Engelbreth-Holm-Swarm (EHS) Mouse Tumor FORMULATION: Dulbecco's Modified Eagle's Medium with 50 µg/mL gentamycin.

Corning Matrigel Basement Membrane Matrix is compatible with all culture media.

STORAGE: Stable when stored at -20°C. Freeze thaws should be minimized by aliquotting into one

–  –  –

COATING PROCEDURES:

Corning Matrigel Basement Membrane Matrix may be used in several ways. The Thin Gel Method is useful for plating cells on top of the gel, the Thick Gel Method allows you to grow cells within a three dimensional matrix, and the Thin Coating Method (no gel) provides you with a complex protein layer on top of which to grow your cells. Make your selection based on the final result that you wish to achieve, whether it is cell growth, attachment or differentiation.

NOTE: Application specific protocols are posted on the support web page.* The protein concentration for Corning Matrigel Matrix products is lot specific and provided on the Certificate of Analysis. For consistent results dilute Corning Matrigel Matrix products by calculating the specific protein concentration (mg/mL) required. To maintain a gelled consistency we recommend not diluting Corning Matrigel Matrix to less than 3 mg/mL. Use ice-cold serum-free medium to dilute Corning Matrigel Matrix. Mix by pipetting up and down or by swirling the vial in ice.

Thin Gel Method

1. Thaw Corning Matrigel Basement Membrane Matrix as recommended. Using cooled pipets, mix the Corning Matrigel Basement Membrane Matrix to homogeneity.

2. Keeping culture plates on ice, add 50 µl/cm2 of growth surface.

3. Place plates at 37oC for 30 minutes.

4. If necessary aspirate unbound material just before use and rinse gently using serum-free medium. Ensure that the tip of the pipet does not scratch the coated surface. Plates are now ready to use.

Thick Gel Method

1. Thaw Corning Matrigel Basement Membrane Matrix as recommended. Using cooled pipets, mix the Corning Matrigel Basement Membrane Matrix to homogeneity.

2. Keep culture plates on ice. Add cells to Corning Matrigel Basement Membrane Matrix and suspend using cooled pipets. Add 150-200 µl/cm2 of growth surface.

Place plates at 37oC for 30 minutes. Culture medium may now be added. Cells may also be cultured on top of this 3.

gel.

Thin Coating Method

1. Thaw Corning Matrigel Basement Membrane Matrix as recommended. Using cooled pipets, mix the Corning Matrigel Basement Membrane Matrix to homogeneity.

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3. Add diluted Corning Matrigel Basement Membrane Matrix to vessel being coated. Quantity should be sufficient to cover entire growth surface easily. Incubate at room temperature for one hour.

4. Aspirate unbound material and rinse gently using serum-free medium. Plates are now ready to use.

CELL RECOVERY:

Corning Dispase (Cat. No. 354235), Corning Cell Recovery Solution (Cat. No. 354253).

Most efficient recovery of cells growing on Corning Matrigel Basement Membrane Matrix is accomplished using Corning Cell Recovery Solution that depolymerizes the Corning Matrigel Basement Membrane Matrix within 7 hours on ice or with Corning Dispase, a metalloenzyme which gently releases the cells allowing for continuous culture.





*NOTE: For technical resources contact Technical Support at:

tel: 800.492.1110; email: CLSTechServ@corning.com

REFERENCES:

1. Kleinman HK, et al, Isolation and characterization of type IV procollagen, laminin, and heparan sulfate proteoglycan from the EHS sarcoma, Biochemistry 21:6188 (1982).

2. Kleinman HK, et al, Basement membrane complexes with biological activity, Biochemistry 25:312 (1986).

3. Vukicevic S, et al, Identification of multiple active growth factors in basement membrane Matrigel suggests caution in interpretation of cellular activity related to extracellular activity related to extracellular matrix components, Exp Cell Res 202:1 (1992).

4. McGuire PG and Seeds NW, The interaction of plasminogen activator with a reconstituted basement membrane matrix and extracellular macromolecules produced by cultured epithelial cells, J. Cell. Biochem. 40:215 (1989).

5. Biederer T and Scheiffele P, Mixed-culture assays for analyzing neuronal synapse formation, Nat Protoc 2(3):670 (2007).

6. Li Y, et al, Essential Role of TRPC channels in the guidance of nerve growth cones by brain-derived neurotrophic factor, Nature 434:894 (2005).

7. Bi Y, et al, Use of cryopreserved human hepatocytes in sandwich culture to measure hepatobiliary transport, Drug Metab Dispos. 34(9):1658 (2006).

8. Gassei K, et al, Immature rat seminiferous tubules reconstructed in vitro express markers of Sertoli cell maturation after xenografting into nude mouse hosts, Mol Hum Reprod. 16(2):97 (2010).

9. Yu X, et al, Essential role of extracellular matrix (ECM) overlay in establishing the functional integrity of primary neonatal rat sertoli cell/gonocyte co-cultures: An improved in vitro model for assessment of male reproductive toxicity, Toxicol Sci 84(2):378 (2005).

10. Chandrasekher G, and Sailaja D, Differential activation of phosphatidylinositol 3-kinase signaling during proliferation and differentiation of lens epithelial cells, Invest Ophthalmol Vis Sci. 44(10):4400 (2003).

11. McGuire PG, and Orkin RW, A simple procedure to culture and passage endothelial cells from large vessels of small animals, Biotechniques 5(6):456 (1987).

12. Bissel DM, et al, Support of cultured hepatocytes by a laminin-rich gel. Evidence for a functionally significant subendothelial matrix in normal rat liver, J. Clin Invest. 79:801 (1987).

13. Page JL, et al, Gene expression profiling of extracellular matrix as an effector of human hepatocyte phenotype in primary cell culture, Toxicol Sci 97(2):384 (2007).

14. Cooley LS, et al, Reversible transdifferentiation of blood vascular endothelial cells to a lymphatic-like phenotype in vitro, J Cell Sci. 123(Pt 21):3808 (2010).

15 Li ML, et al, Influence of a reconstituted basement membrane and its components on casein gene expression and secretion in mouse mammary epithelial cells, Proc. Nat. Acad. Sci. USA 84:136 (1987).

16 Barcellof MH, et al, Functional differentiation and aveolar morphogenesis of primary mammary cultures on reconstituted basement membrane, Development 105:223 (1989).

17. Roskelley CD, et al, Extracellular matrix-dependent tissue-specific gene expression in mammary epithelial cells requires both physical and biochemical signal transduction, Proc. Nat. Acad. Sci. USA 91(26):12378 (1994).

18. Xu R, et al, Extracellular matrix-regulated gene expression requires cooperation of SWI/SNF and transcription factors, J. Biol. Chem. 282(20):14992 (2007).

19. Debnath J, et al, Morphogenesis and oncogenesis of MCF-10A mammary epithelial acini grown in three-dimensional basement membrane cultures, Methods 30(3):256 (2003).

20. Muthuswamy SK, et al, ErbB2, but not ErbB1, reinitiates proliferation and induces luminal repopulation in epithelial acini, Nat. Cell Biol. 3(9):785 (2001).

21. Albini A, et al, A rapid in vitro assay for quantitating the invasive potential of tumor cells, Cancer Res,47:3239 (1987).

22. Poincloux R, et al, Contractility of the cell rear drives invasion of breast tumor cells in 3D Matrigel, Proc Natl Acad Sci USA.108(5):1943 (2011).

23. Madison R, et al, Increased rate of peripheral nerve regeneration using bioresorbable nerve guides and laminin containing gel, Exp. Neurology 88:767 (1985).

24. Xu XM, et al, Axonal regeneration into Schwann cell-seeded guidance channels grafted into transected adult rat spinal cord, J. Comp. Neurol.

351(1):145 (1994).

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SPC-356234 Rev 6.0 -3Lopatina T, et al, Adipose-derived stem cells stimulate regeneration of peripheral nerves: BDNF secreted by these cells promotes nerve healing and axon growth de novo, PLoS One 6(3):e17899 (2011).

26. Kubota Y, et al, Role of laminin and basement membrane in the morphological differentiation of human endothelial cells into capillary-like structures, J.

Cell Biol. 107:1589 (1988).

27. Ponce ML, Tube formation: an in vitro matrigel angiogenesis assay, Methods Mol Biol. 467:183 (2009).

28. Passaniti A, et al, A simple, quantitative method for assessing angiogenesis and anti-angiogenic agents using reconstituted basement membrane, heparin, and fibroblast growth factor, Lab Invest. 67:519 (1992).

29. Isaji M, et al, Tranilast inhibits the proliferation, chemotaxis and tube formation of human microvascular endothelial cells in vitro and angiogenesis in vivo, Br J Pharmacol 122:1061 (1997).

30. Adini A, et al, Matrigel cytometry: a novel method for quantifying angiogenesis in vivo, J Immunol Method. 342(1-2):78 (2009).

31. Albini A, et al, Matrigel promotes retinoblastoma cell growth in vitro and in vivo, Int. J. Cancer 52(2):234 (1992).

32. Yue W, and Brodie A, MCF-7 human breast carcinomas in nude mice as a model for evaluating aromatase inhibitors, J. Steroid Biochem. Mol. Biol.

44(4-6):671 (1993).

33. Angelucci A, et al, Suppression of EGF-R signaling reduces the incidence of prostate cancer metastasis in nude mice, Endocr-Relat Cancer 13(1):197 (2006).

34. Moraes RC, et al, Constitutive activation of smoothened (SMO) in mammary glands of transgenic mice leads to increased proliferation, altered differentiation and ductal dysplasia, Development 134:1231 (2007).

35. Zeng YA, and Nusse R, Wnt proteins are self-renewal factors for mammary stem cells and promote their long-term expansion in culture, Cell Stem Cell 6:568 (2010).

36. Jeselsohn R, et al, Cyclin D1 kinase activity is required for the self-renewal of mammary stem and progenitor cells that are targets of MMTVErbB2 tumorigenesis, Cancer Cell 17:65 (2010).

37. Quintana E, et al, Efficient tumor formation by single human melanoma cells, Nature 456:593 (2008).

CALIFORNIA PROPOSITION 65 NOTICE

WARNING: This product contains a chemical known to the state of California to cause cancer.

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SPC-356234 Rev 6.0 -4-



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