Ccl25

The human chemokines CCL1, CCL5, CCL7, CCL11, CCL17, CCL19, CCL20, CCL25, CCL27, CXCL8, CXCL11, CXCL12, CXCL13, CXCL16, and CX₃CL1 were purchased from PeproTech, whereas XCL1 was purchased from R&D Systems. All chemokines were reconstituted in a buffer containing 0.1% (w/v) bovine serum albumin (BSA) and 1 mM acetic acid.

A micromass pellet system was used to determine a factor enhancing AF matrix formation. After the third passage, AF cells of three individual donors were washed twice with serum-free differentiation medium (Dulbeccos modified Eagle Medium (4.5 g/L glucose) (Biochrom), 2% HEPES (Biochrom), 1% penicillin/streptomycin (Biochrom), 1% ITS⁺ supplement (Sigma-Aldrich), 0.1 µM dexamethasone (Sigma-Aldrich), 1 mM sodium pyruvate (Sigma-Aldrich), 0.17 mM ascorbic acid (Sigma-Aldrich), 0.35 mM proline (Sigma-Aldrich)) and 250,000 cells were transferred to 15 mL tubes. To the serum-free medium BMP2, BMP7, BMP12 (growth differentiation factor 7 (GDF7)), BMP14 (GDF5), TGFβ3 or CCL25 (all Peprotech) were added in concentrations of 10, 50 or 100 ng/mL. Additionally, samples with 2.5% PRP and non-stimulated controls without any other factor were prepared. Six samples of each factor and each concentration were prepared. Over all, 120 pellet cultures in 20 different media were prepared for cells of each donor. The pellets were cultivated for 28 days with a medium change three times a week. Samples were taken at day 2, day 14, and day 28. Each pellet was placed in a cryomold and embedded in TissueTek (Sakura Finetek). After freezing in liquid nitrogen the pellets were stored at −80 °C until used for histological and immunohistochemical analysis.

The binding affinity of recombinant CrmD for mouse chemokines (Peprotech Inc., London, UK) and the ability of wild type CrmD and mutant N77F to interact with mouse TNF (R&D Systems, Minneapolis, USA) and Ccl25 (Peprotech Inc., London, UK) were determined by SPR using a Biacore X biosensor (GE Healthcare).
For affinity determinations, recombinant CrmD was immobilized onto a flow cell of a CM4 chip at low density (900 RUs) by the amine coupling protocol. One flow cell was left empty to be used as reference. Increasing concentrations of mouse chemokines were injected in HBS-EP buffer (GE Healthcare) over the chip surface and their binding was recorded for 120 s followed by a 300 s dissociation period. Surface was regenerated with glycin-HCl pH2.0 between injections. Binding sensorgrams were analyzed by the BiaEvaluation software (GE Healthcare) and fitted to a general 1:1 Langmuir binding model.
For binding assays, recombinant wild type and N77F CrmD proteins were immobilized on to a flow cell of a CM4 chip at high density (1500 RUs) as explained above. 100 nM of mouse TNF or mouse Ccl25 were injected over the chip in HBS-EP buffer and their association was monitored during 120 s followed by a 120 s dissociation. Binding sensorgrams were processed and analyzed using the BiaEvaluation software.