Chemerin

Internalization of CMKLR1 upon activation by chemerin was measured by flow cytometry using pro-inflammatory macrophages from WT (control), Grk6−/−, and Barr2−/− mice. Cells were aliquoted (2.5 × 10⁵ cells/ tube), starved for 1 hour in serum-free RPMI 1640, and then stimulated with either 6.25 nM or 200 nM chemerin (R&D systems) for 30 sec, 1 min, 5 min, and 10 min at 37 °C + 5 % CO₂. Cells were not stimulated at the 0 minute time point in order to determine maximum receptor expression before addition of ligand. At each time point, receptor internalization was arrested with 4°C buffer (HBSS + 5% FBS + 2 mM EDTA) and by placing cells immediately on ice. Cells were then stained for CMKLR1 (anti-mouse CMKLR1 PE, eBioscience, 12-7582, clone BZ194, discontinued, or anti-mouse CMKLR1, Miltenyi, 130-106-902, clone REA461) and F4/80 (rat anti-mouse F4/80:APC, AbC serotec, MCA497APCT, clone CI:A3-1) as previously described (51 (link)). CMKLR1 surface expression was measured via flow cytometry by gating on F4/80 positive cells to confirm the myeloid lineage and measuring mean fluorescence intensity (MFI) of CMKLR1. Data was normalized as a percent relative to the MFI at 0 min for each cell type.

Cell migration to chemerin was examined using the Falcon^™ HTS FluoroBlok 96-Multiwell Insert System (3 µm pore-size) (BD Biosciences, Bedford, MA) and pro-inflammatory macrophages from WT (control), Grk6−/−, and Barr2−/− mice obtained as previously described. Cells were starved in RPMI 1640 + 1% BSA at 37°C for one hour and labeled with calcein (485 nm/ 527 nm). Cells were washed and suspended in RPMI 1640 + 1% BSA + 10 mM HEPES, and 1 × 10⁵ cells were loaded into FalconTM HTS FluoroBlok 96-Multiwell Insert System chemotaxis chambers (Corning, NY). Cells were allowed to migrate to RPMI/BSA/HEPES medium alone or to medium with addition of 6.25 nM chemerin (R&D Systems, Minneapolis, MN). Fluorescence was measured at 2 minute intervals over the course of 100 minutes at 37°C using a Fluoroskan Ascent Microplate Fluorometer (Thermo Scientific, Waltham, MA). Data was normalized by subtracting time 0 from each subsequent time point for each condition and graphed as fluorescence vs time for the first 100 minute interval. Analysis was done using linear mixed models and tested for a significant group effect using likelihood ratio test.