Measuring Interactions Between ECM and TGFB-Like Proteins - Methods in Molecular Biology: Extracellular Matrix Protocols - page 230

labeling period of only a few hours were found to be sufficient with a particular
cell type, it may be preferable to use media that is totally free of cysteine for
labeling.

2. An incubation with 50 m M ammonium acetate, pH 9.0 can be used following the

lysis step to remove nuclear matrix proteins (as recommended in the procedure
described by Jones et al. (18) . However, owing to the alkaline pH of this buffer,
it is not recommended for analysis of TGF β , because TGF β can be activated by

pH extremes.

3.

35 S-labeled ECM can be used directly without drying, however, it is our experi-
ence that if the matrix is dried first, there are less problems with loss of adherence
later on when the cells are seeded onto the matrix.

4. Any cell type could be used to examine its ability to release LTBP1/latent TGF β

from the labeled ECM at this step. The seeding densities and culture periods
should be optimized for each cell line to determine the conditions for maximal
release of LTBP1/latent TGF from the labeled ECM. We have found the opti-

mum seeding density for avian osteoclast precursors to be 3

β

10 6 cells per well.
This is because in order to form mature osteoclasts, these precursor cells must
first fuse in culture. A higher density of cells allows more efficient fusion. In
avian osteoclast cultures, maximal release of LTBP1/latent TGF

×

is seen on days
3-4. This corresponds with the appearance of mature multinucleated (fused)
osteoclasts. We have found that a good seeding density for MDA-MB-231 human
breast cancer cells is 5

β

10 5 cells/well and that maximal release of LTBP1/latent

×

TGF β from FRC matrix by these cells occurs by 24 h.

5. It is extremely important when preparing cells for these experiments to ensure

that any trypsin or other enzymes that have been used to prepare the cell suspen-
sion be removed prior to seeding the cells onto the matrix. It is recommended that
cell preparations which have been trypsinized be treated with one-tenth volume
of serum to inactivate the enzymes. Additionally the cell suspension should be
washed two times in PBS before plating them onto the labeled matrix.

6. The ECM produced by long-term FRC cultures is usually present as a thick mem-

brane-like layer which is still adherent to the culture plate. If ECMs from other
cell types are used and are found not to adhere very well to the culture plate, the
entire lysate, including insoluble (matrix) material can be transferred to a 1.5 mL
tube. This can then be microfuged and the DOC insoluble pellet washed and used
for plasmin digestion.

7. If a high level of background bands is obtained, even in the nonimmune control

samples, this background can be reduced by using preadsorbed protein G agarose
at this step. This is prepared by incubating the protein G agarose in unlabeled cell
lysate from the same cells as those used in the immunoprecipitation (this blocks
nonspecific binding sites on the protein G agarose with unlabeled protein and
thereby reduces background caused by nonspecific adsorption of irrelevant pro-
teins). For 1 mL agarose beads (bed volume) incubate with 4 mL unlabeled cell
lysate for 1 h at room temperature on a rotator. Centrifuge the protein G agarose,
aspirate the lysate, then wash agarose four times in PBS. Resuspended agarose in
PBS to give a 50% slurry.