Matrigel preparation from EHS cells

1. Obtain Englebreth-Holm-Swarm cells (EHS, ATCC CRL-2108). This is a murine sarcoma cell line that is serially propagated in syngeneic mice (C57BL6).

2. Thaw ampoule or vial and inject IM into the hind limb (Biceps Femoris ?) of 2 mice.

3. Allow to grow. Within 6-8 weeks most of the upper limb muscle mass will be replaced

by tumor causing the leg to extend unnaturally.

4. Sacrifice animals and aseptically excise tumor mass. Macerate (can use a sterile Polytron homogenized set on low speed). Inject more animals. Generally the tumors from 2 mice will provide enough material to inject 30-40 mice into 1 hind limb each.

5. After tumors develop (6-8 weeks) use the following procedure from example 1 of U.S.

patent 4,829,000: Based on 100g tumor tissue freshly excised from sacrificed animals. All steps to be conducted at 4° C unless otherwise noted.

1. Homogenize macerated tumor tissue in 200 ml of sterile high salt buffer (3.4M NaCl, 50 mM Tris, 4 mM EDTA, 2 mM NEM (N-ethylmaleimide) pH 7.4). I used a Polytron homogenizer.

2. Centrifuge 12,000 x g for 15 min. Discard supernatant. 3. Homogenize pellet in 200 ml of the high salt buffer. 4. Centrifuge 12,000 x g for 15 min. Discard supernatant. 5. Repeat steps 3&4 6. Homogenize pellet in 100 ml of urea buffer (2M urea, 50 mM Tris, 0.15 M NaCl, pH

7.4). Stir overnight in the cold. 7. Centrifuge 18,000 x g for 20 min. Save supernatant on ice. 8. Homogenize pellet in 50 ml of the above urea buffer. 9. Centrifuge 18,000 x g for 20 min. Combine supernatants. 10. Dialyze against 1 liter of cold dialysis buffer (50 mM Tris, 0.15 M NaCl pH 7.4)

containing 5 mL of chloroform to sterile the matrigel. 4-8h. I used 10 kDa cutoff Spectrapore tubing.

11. Change to dialysis buffer without CHCl3 4-8h. Repeat 2 times. 12. Dialyze against DMEM overnight. 13. Sterilize outside of dialysis membrane with ethanol and transfer matrigel to sterile tubes.

Freeze at -80º C. Thaw and polymerize by warming to 24-37° C.