Methods for Preparing Extracellular Matrix and Quantifying Insulin-like Growth Factor-Binding Protein Binding to the ECM - Methods in Molecular Biology: Extracellular Matrix Protocols - page 213

6. Tris-buffered saline containing 3% BSA.
7. IGFBP-5.
8. Iodobeads (Pierce, Rockford, IL).
9. 0.25 M NA 2 PO 4 , pH 7.2.
10. Sephadex G-100.
11. Gamma spectrometer.

2.3. Procedures to Verify that the ECM has been Extracted
1. Biomax membranes (Milipore).

2. Glycerol.

3. SDS-PAGE equipment.

4. Vitronectin.

5. Anti-vitronectin antiserum (Sigma).

2.4. Preparation of Fibroblast (GM-10) ECM
1. Human dermal fibroblasts (GM-10) from Coriell Institute (Camden, NJ).

2. Primaria 24-well plates (Falcon Plastics, Becton Dickinson, Rutherford, NJ).

3. Lysis buffer: 0.5% triton X-100 in PBS, 10 m M EDTA, pH 7.4.

4. 25 m M ammonium acetate, pH 9.0.

5. 1 M HEPES, pH 7.3.

6. 30 m M sodium phosphate buffer with 1% BSA, pH 7.4.

2.5. Binding of 125 I-IGFBP-5 on Unlabeled IGFBP-5
to Fibroblast ECM
1. IGFBP-5.

2. 30 m M sodium phosphate buffer, 0.1% BSA, pH 7.4.

3. 0.3 N NaOM.

4. Immobilon membranes.

3. Methods
3.1. Preparation of Porcine Smooth Muscle Cell ECM
pSMCs do not synthesize abundant ECM; therefore, the ECM cannot be
seen under the microscope. Extracting the ECM is thus a difficult and delicate
procedure. Bear in mind that ECM does not remain adherent to culture plates
for more than a few weeks, even in optimal storage. For more information
about the properties of pSMC ECM, see Note 1 .

1. Porcine aortic smooth muscle cells prepared from 3-wk-old pigs as described by

Ross (9) .

2. Porcine smooth muscle cells (pSMC) in DMEM-H supplemented with 10% FBS,

penicillin at 100 U/mL, and streptomycin at 100 µ g/mL (Life Technologies).

3. Subculture the cells in 10-cm plates (Falcon #3001) until confluent.

4. Grow pSMC in 10-cm culture dishes (Falcon 3001) until they are densely

confluent. Change the media the day before the ECM is prepared.