Preparation of Basement Membrane Components from EHS Tumors

Hynda K. Kleinman1

1 National Institute of Dental Research/NIH, Bethesda, Maryland
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 10.2
DOI:  10.1002/0471143030.cb1002s00
Online Posting Date:  May, 2001
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Abstract

This unit describes methods for passaging and harvesting the basement membrane matrix‐producing EHS tumor and for the subsequent isolation of a crude mixture of basement membrane components termed Matrigel, which promotes the differentiation of a variety of epithelial, endothelial, and neuronal cells. Procedures for the isolation of the adhesive glycoprotein laminin‐1 and of type IV collagen are also included. Support protocols cover the maintenance and harvesting of EHS tumors in mice and maintenance of mice on a lathrogenic diet.

     
 
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Table of Contents

  • Basic Protocol 1: Preparation of Matrigel
  • Basic Protocol 2: Preparation of Laminin‐1
  • Basic Protocol 3: Preparation of Type IV Collagen
  • Support Protocol 1: Maintenance and Harvest of EHS Tumors
  • Support Protocol 2: Maintenance of Mice on a Lathrogenic Diet
  • Reagents and Solutions
  • Commentary
  • Literature Cited
     
 
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Materials

Basic Protocol 1: Preparation of Matrigel

  Materials
  • EHS tumor (see protocol 4)
  • 3.4 M NaCl buffer (see recipe), 4°C
  • 2 M urea buffer (see recipe), 4°C
  • Tris‐buffered saline (TBS; appendix 2A),4°C
  • Chloroform
  • Tissue culture medium (e.g., DMEM, RPMI)
  • 70% ethanol
  • Electric homogenizer (e.g., Polytron)
  • Centrifuge and rotor, 4°C
  • Sterile hemostat
  • Additional materials and equipment for dialysis and protein assays (see appendix 3A)
NOTE: All reagents and equipment should be prechilled to 4°C, and all procedures must be performed at 4°C.

Basic Protocol 2: Preparation of Laminin‐1

  Materials
  • EHS tumor (see protocol 4)
  • 3.4 M NaCl buffer (see recipe), 4°C
  • 0.5 M NaCl buffer (see recipe), 4°C
  • Ammonium sulfate
  • TBS ( appendix 2A), 4°C
  • NaCl
  • Chloroform
  • Electric homogenizer (e.g., Polytron)
  • Centrifuge and rotor, 4°C
  • Sterile hemostat
  • Additional materials and equipment for dialysis and protein assays (see appendix 3A)
NOTE: All reagents and equipment should be prechilled to 4°C, and all procedures must be performed at 4°C.

Basic Protocol 3: Preparation of Type IV Collagen

  Materials
  • Tumor homogenate (see protocol 2, step ), prepared from animals fed a lathrogenic diet (see protocol 5)
  • 0.5 M NaCl buffer (see recipe), 4°C
  • 2.0 M guanidine⋅HCl buffer (see recipe), 4°C
  • 2.0 M guanidine⋅HCl buffer containing 32 mg/liter dithiothreitol (DTT), 4°C
  • 0.5 M acetic acid (28.5 ml acetic acid/liter water), 4°C
  • Centrifuge and rotor, 4°C
  • Additional materials and equipment for dialysis and protein assays (see appendix 3A)
NOTE: All reagents and equipment should be prechilled to 4°C, and all procedures must be performed at 4°C.

Support Protocol 1: Maintenance and Harvest of EHS Tumors

  Materials
  • Fresh EHS tumor
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • PBS containing 100 U/ml penicillin and 100 µg/ml streptomycin
  • C57BL/6 mice
  • Anesthetic (e.g., 3.2% Avertin)
  • Dimethylsulfoxide (DMSO)
  • 70% ethanol
  • 50‐ml plastic tubes
  • 20‐ to 30‐ml and 3‐ml syringes
  • 16‐G needles
  • Centrifuge and rotor, 4°C
NOTE: All protocols using live animals must first be reviewed and approved by an Institutional Animal Care and Use Committee (IACUC) and must follow officially approved procedures for the care and use of laboratory animals.

Support Protocol 2: Maintenance of Mice on a Lathrogenic Diet

  Materials
  • Lathrogenic chow (see recipe)
CAUTION: Lathrogenic chow contains toxic materials; gloves, mask, and lab coat should be worn when handling it.
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Figures

Videos

Literature Cited

Literature Cited
   Albini, A., Iwamoto, Y., Kleinman, H.K., Martin, G.R., Kozlowski, J.M., and McEwan, R.N. 1987. A rapid in vitro assay for quantitating the invasive potential of tumor cells. Cancer Res. 47:3239‐3245.
   Beck, K., Hunter, I., and Engel, J. 1990. Structure and function of laminin: Anatomy of a multidomain protein. FASEB J. 4:148‐160.
   Burgeson, R.E., Chiquet, M., Deutzmann, R., Ekblom, P., Engel, J., Kleinman, H.K., Martin, G.R., Meneguzzi, G., Paulsson, M., Sanes, J., Timpl, R., Tryggvason, K., Yamada, Y., and Yurchenco, P.S. 1994. A new nomenclature for the laminins. Matrix Biol. 14:209‐211.
   Carey, D.J., Todd, M.S., and Rafferty, C.M. 1986. Schwann cell myelination: Induction by exogenous basement membrane–like extracellular matrix. J. Cell Biol. 102:2254‐2263.
   Evercooren, A.B., Kleinman, H.K., Ohno, S., Schwartz, J.P., and Dubois‐Dalqc, M. 1982. Factors promoting neurite growth in human fetal sensory ganglia cultures. J. Neurosci. Res. 8:179‐194.
   Fridman, R., Kibbey, M.C., Royce, L.S., Zain, M., Sweeney, T.M., Jicha, D.L., Yannelli, J.R., Martin, G.R., and Kleinman, H.K. 1991. Basement membrane (Matrigel) enhances both the incidence and growth of subcutaneously injected human and murine cells. J. Natl. Cancer Inst. 83:769‐774.
   Hadley, M.A., Byers, S.W., Suarez‐Quian, C.A., Kleinman, H.K., and Dym, M. 1985. Extracellular matrix regulates Sertoli cell differentiation, testicular cord formation, and germ development. J. Cell Biol. 101:1511‐1522.
   Kleinman, H.K., McGarvey, M.L., Liotta, L.A., Gehron Robey, P., Tryggvason, K., and Martin, G.R. 1982. Isolation and characterization of native type IV collagen from the EHS sarcoma. Biochemistry 24:6188‐6193.
   Kleinman, H.K., McGarvey, M.L., Hassell, J.R., Star, V.L., Cannon, F.B., Laurie, G.W., and Martin, G.R. 1986. Basement membrane complexes with biological activity. Biochemistry 25:312‐318.
   Kleinman, H.K., Kibbey, M.C., Schnaper, W.H., Yamamura, K., Weeks, B.S., and Grant, D.S. 1993. The laminins: A family of basement membrane glycoproteins important in cell differentiation and tumor metastasis. Vitamins Hormones 47:161‐186.
   Kubota, Y., Kleinman, H.K., Martin, G.R., and Lawley, T.J. 1988. Role of laminin and basement membrane in the differentiation of human endothelial cells into capillary‐like structures. J. Cell Biol. 107:1589‐1597.
   Li, L., Aggeler, M.J., Farson, D.A., Hatier, C., Hassell, J.R., and Bissell, M.J. 1986. Influence of a reconstituted basement membrane and its components on casein gene expression and secretion in mouse mammary epithelial cells. Proc. Natl. Acad. Sci. U.S.A. 84:136‐140.
   Martin, G.R. and Timpl, R. 1987. Laminin and other basement membrane components. Annu. Rev. Cell Biol. 3:57‐85.
   Orkin, R.W., Gehron, P., McGoodwin, E.B., Martin, G.R., Valentine, T., and Swarm, R. 1977. A murine tumor producing a matrix of basement membrane. J. Exp. Med. 145:204‐220.
   Passaniti, A., Taylor, R.M., Pili, R., Guo, Y., Long, P.V., Haney, J.A., Pauly, R.R., Grant, D.S., and Martin, G.R. 1992. A simple, quantitative method for assessing angiogenesis and antiangiogenic agents using reconstituted basement membrane, heparin and fibroblast growth factor. Lab. Invest. 67:519‐528.
   Timpl, R. and Brown, J.C. 1994. Supramolecular assembly of basement membranes. BioEssays 18:123‐132.
   Timpl, R., Rohde, H., Robey, P.G., Rennard, S.I., Foidart, J.M., and Martin, G.R. 1979. Laminin—A glycoprotein from basement membrane. J. Biol. Chem. 254:9933‐9939.
   Yurchenco, P.D. and Schittny, J.C. 1990. Molecular architecture of basement membrane. FASEB J. 4:1577‐1590.
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