Chemokine receptor trafficking in HEK293 cells

HEK293^WT; HEK293 β-arrestin 1^KO; β-arrestin 2^KO; β-arrestin 1, 2^KO cells transfected with receptors (and GFP in a T2A system or with an mGPR182-venus construct), were grown on MatTek slides. These were incubated with 100 nM chemokines (all labeled in AF647, ATTO565 or ATTO700) for 45 minutes at 37°C. Cells were washed with PBS, fixed in 4% paraformaldehyde (PFA), permeabilized, stained with antibodies, and mounted in either Fluoromount (Sigma, Cat: F4680) or DAPI-containing Duolink in situ mounting medium (Sigma, Cat: DUO82040). Membranes were visualized either by staining with MemBrite Fix Cell Surface Staining Kit (Biotium) following manufacturer’s instructions, or by transiently transfecting cells with a plasmid expressing the myristoylation/palmitylation motif from the Lck fused to the N-terminus of mCherry generating a plasma membrane marker (13 (link)).
For live imaging experiments, HEK293 expressing mGPR182 GFP were grown on MatTek slides. On the day of the experiment, complete medium was replaced with Optimem (Gibco) containing 10% FBS. CCL20-AF647 was added dropwise during live acquisition at t = 1 minute, at a final concentration of 200 nM. Live imaging was performed using a Leica SP5 confocal microscope. Chemokine uptake was quantified using ImageJ by measuring Integrated Density (set to threshold) on selected ROIs.