Cells were incubated in DMEM, containing 4 mM HEPES and 1% BSA, in the presence or in the absence of 1 nM of chemerin (R&D) or CCL19 (Peprotech, Rocky Hill, NJ, USA) for the indicated times. Where indicated, cells were preincubated with 80 µm Dynasore (dynamin inhibitor, Sigma, St. Louis, MO, USA). After incubation, residual ligand in the supernatant was quantified by ELISA (DY2324 and DY361, R&D).
To evaluate chemerin into the cells, cells were incubated as previously described, in the absence or in the presence of 1 nM chemerin at 37°C for 2 h. Then plates were put in ice and rinsed with PBS. Cells were washed with different buffers for 5 min at room temperature: PBS, acidic buffer [150 mM NaCl, 100 mM glycine, pH = 3, in water (32 (link))], or acidic followed by high salt buffer (2 M NaCl in water). After rinsing with PBS, cells were lysed for 30 min in ice (1% Triton X-100, 5 mM EDTA, in PBS), then centrifuged (16,000g) at 4°C for 15 min. Internalized chemerin was evaluated by ELISA from supernatants obtained after centrifugation. The quantity of chemerin was normalized over total protein content, quantified using Bradford reagent (Biorad, Hercules, CA, USA).
To evaluate chemerin into the cells, cells were incubated as previously described, in the absence or in the presence of 1 nM chemerin at 37°C for 2 h. Then plates were put in ice and rinsed with PBS. Cells were washed with different buffers for 5 min at room temperature: PBS, acidic buffer [150 mM NaCl, 100 mM glycine, pH = 3, in water (32 (link))], or acidic followed by high salt buffer (2 M NaCl in water). After rinsing with PBS, cells were lysed for 30 min in ice (1% Triton X-100, 5 mM EDTA, in PBS), then centrifuged (16,000g) at 4°C for 15 min. Internalized chemerin was evaluated by ELISA from supernatants obtained after centrifugation. The quantity of chemerin was normalized over total protein content, quantified using Bradford reagent (Biorad, Hercules, CA, USA).
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