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Preparing Samples

Note: Samples cannot be stored or returned after testing is complete. To limit number of freeze-thaws, preactivate fresh samples before freezing.

CULTURE SUPERNATANTS, SERUM OR PLASMA:

The laboratory is located in 500 square feet of high quality laboratory space in the MSTF research building located on the campus of the University of Maryland, Baltimore. The CCL offers assay services to both University of Maryland investigators and extramural investigators.

CELL LYSATES:

Collect cells in a balanced salt solution containing some protein to decrease binding of cytokines of interest to plastic wells and tubes. Culture medium or PBS contatining 1 to 10% serum or 2% BSA works fine. If available, lyse cells using a cell sonicator with three 5 to 10 second bursts, chilling on ice between sonications. Alternatively, freeze thaw with three cycles. If cells are associated,you may need to add a low concentration detergent(1% Triton X100). Remove cell debris by high speed 3 min centrifugation in a microcentrifuge. Protease inhibitors may help prevent loss of cytokine protein in some cell lysates. If so, we suggest adding 4mM EDTA, 1mM Phenylmethylsulfonyl fluoride(PMSF) and 1 ug/ml leupeptin to your lysis buffer.

TRANSFORMING GROWTH FACTOR BETA ACTIVATION:

TGF beta is synthesized as an inactive precursor which is cleaved producing the active growth factor bound to a neutralized peptide. The peptide must be dissociated to activate the TGF beta. This is accomplished in vitro by the acidification protocol that follows. Pre-activation of body fluids is required to measure total TGF beta levels, but performing the ELISA without activation will measure TGF beta that is active in vivo. Whether you preactivate body fluids or not depends on the question you are asking. TGF beta in culture supernatants is inactivate and should be activated prior to assay.

TGF BETA PREACTIVATION PROTOCOL:

TCell culture supernatants: To 1 ml cell culture supernatant, add 0.2 ml 1N HCl, mix well, and incubate at room temperature for 10 min. Neutralize the acidified sample by adding 0.2 ml 1.2N NaOH/0.5M HEPES, mix well and assay (correct for 1:4 dilution).

Serum/Plasma: To 0.5 ml serum/plasma, add 0.5 ml 2.5N acetic acid / 10N urea, mix well and incubate at room temperature for 10 min. Neutralize the acidified sample by adding 0.5 ml 2.7 N NaOH/1.0M HEPES, mix well and assay(correct for 1:30 dilution).