Radioactive Glycosaminoglycan Labeling Protocol

1. Label cells with 50 mCi/ml [35S]O4 (t1/2 35S=87.4 days) in F12 + 10% dialyzed FBS (+ 100 Units Penicillin, but leave out the streptomycin). Other antibiotics can be added, but make sure they are not the sulfate salts. If glucosamine labeling are desired, use 20 Ci/ml [6-3H]glucosamine in F12 defined medium containing dialyzed FBS and 1 mM glucose. Remove 5 L and dilute to 50, count 10 L to determine actual radiospecific activity of medium.
2. Culture the cells for 2-3 days

The following is set up for 24 well plates, 0.5 ml of medium per well. Scale as needed, except where indicated.

3. GAG collection

Media GAGs: Remove media, centrifuge to remove floating cells, and save supernate.

Cell surface GAGS: Rinse the monolayer 3X with PBS (discard rinses), trypsin-treat the cells, spin cells out and collect trypsinate (supernatant solution). If you want to normalize to protein, solubilize cells in 0.15 ml 0.1 N NaOH, @ RT 20. Remove 10 and 25 ml for BioRad protein analysis.

Intracellular GAGs: Use the cell pellet. Solubilize cells in 0.15 ml 0.1 N NaOH, @ RT 20. Remove 10 and 25 ml for BioRad protein analysis.

Total Cell GAGs. Rinse the monolayer 3X with PBS (discard rinses). Solubilize cells in 0.15 ml 0.1 N NaOH, @ RT 20. Remove 10 and 25 ml for BioRad protein analysis.