Method

1 Place 20 x 10G purified human monocytes in 15 ml polypropylene tubes in 1 ml of RPMI1640 medium.

2 Add appropriate stimuli {e.g. 50 ng/ml PMA or 10~7 M fMLP dissolved in RPMI1640 medium) and incubate at 37°C for 20 mln.

3 Recover medium by pipette.

4 Concentrate medium ten times by membrane filtration using appropriate molecular weight.

5 Add 100 n-1 of concentrate medium to 1 nM 12SI-iabelled ligand (e.g. IL-lfi) in 1.5 ml Eppendorf tube and incubate for 4 h at 4 °C.

6 Add 1 nM disuccinimydilsuberate (DSS) and incubate at 4°C for 30 min.

7 Separate samples on 8% SDS-PAGE under reducing conditions.

8 Expose dried gels to photographic film for one to three days, as needed.

9 Process films for autoradiography using standard methods.

Soluble cytokine receptors released by mononuclear phagocytes either con-stitutively or after exposure to a variety of signals can be assessed conveniently by cross-linking using this method. Alternative detection methods for cell-free receptors include Western blotting or ELISA. The latter is the approach of choice to measure soluble cytokine receptors in body fluids. Cross-linking is a simple and straightforward approach with high sensitivity which allows recognition of soluble receptors for which a sensitive ELISA is not available. Moreover, it provides direct evidence that the soluble receptor can recognize the appropriate ligand. Using cold ligand, the specificity and relative affinity of the soluble receptor can also be evaluated (26).

References

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22. Colotta, F., Orlando, S„ Fadlon, E. J„ Sozzani, S„ Matteucci, C, and Mantovani, A. (1995) J. Exp. Med., 181, 2181.

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Chapter 6

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