Measuring Interactions Between ECM and TGFB-Like Proteins - Methods in Molecular Biology: Extracellular Matrix Protocols - page 227

11. Gel fixing solution 1 (20% TCA, 50% isopropanol): to 30 mL distilled water, add

20 mL 100% trichloroacetic acid (TCA) solution (100 g TCA made up to 100 mL
with distilled water, stable for several mo), add 50 mL isopropanol. This solution
should be made fresh on the day of use.

12. Gel fixing solution 2 (10% TCA, 25% isopropanol): make up gel fixing solution

1 and dilute 1:1 with distilled water. This solution should be made fresh on the
day of use.

3. Methods
Note: the procedures for isolation of FRC osteoblasts (11 , 14-15) , and avian
osteoclast precursor cells (16) are well-established techniques and will not be
described in this account. The reader is referred to the relevant publications to
obtain details of these procedures. The procedures described below are modi-
fied from protocols originally described in (9-13) . The reader is referred to
these articles for further information and variations on these techniques.

3.1. Preparation of 35 S-Labeled ECM
1. 10 4 cells/well in 4 mL Plate FRC osteoblasts in a six-well culture plate at 8 ×

-MEM supplemented with 10% FCS, 100 U/mL P/S, 2 m M LG. Culture at
37

α

° C in a 5% CO 2 incubator.

2. When the cells reach confluence (approximately 3 d later) change the media to

4 mL α -MEM supplemented with 5% FCS, 100 U/mL P/S, 2 m M LG, 100 µ g/mL

ascorbic acid, 3 m M β -glycerophosphate.

3. Continue culturing the cells, changing the media every 3 d. Between days 6-12 a

thick layer of ECM is laid down and multilayered “nodules” of cells should be
observed. These then become mineralized, forming bone-like structures.

4. On day 12 of culture, aspirate the media, wash the cells twice in PBS and then

radiolabel the cells for 48 h using 2 mL radiolabeling media containing 100 µ Ci

35 S-cysteine per mL ( see Note 1 ).

5. After labeling, aspirate the media and wash the cells twice in ice-cold PBS. Add

2 mL ice-cold lysis buffer and incubate for 15 min at 4 C. Once the cell mem-

branes are disrupted, this should leave a thick, membrane-like layer of ECM
adherent to the culture plate. Extreme care should be taken with subsequent
washes to avoid disturbing the adherent layer of matrix. Gently aspirate the lysis
buffer and wash the insoluble matrix layer 5 times with 4 mL ice-cold PBS ( see
Note 2 ).

°

6. Aspirate the final PBS wash and air-dry the plates in the sterile laminar flow

hood ( see Note 3 ).

3.2. Analysis of Release of LTBP1 and TGF β

from 35 S-Labeled ECM by Avian Osteoclast Cells
1. Bring 35 S-labeled matrix to room temperature (if stored frozen) and then rehy-

drate for 5 min with 4 mL PBS per well. Discard PBS, wash once more with