Facs fortessa cytometer

U87 cells expressing wild-type or mutant ACKR3 or CXCR4 were distributed into 96-well plates (15 × 10 4 cells per well) and incubated with Alexa Fluor 647-labelled CXCL12 at concentrations ranging from 35 pM to 115 nM for 90 min on ice or for 45 min at 37 °C, respectively. Non-specific binding of CXCL12-AF647 was evaluated on ACKR3-and CXCR4-negative U87 cells and subtracted. For binding competition studies, U87 cells expressing wild-type or mutant ACKR3 or CXCR4 were incubated with CXCL12-AF647 at concentrations equivalent to twice the EC 50 values determined for each ACKR3 variant in saturation binding experiments (Table 1) or 11.5 nM for CXCR4, mixed with unlabelled CXCL12, CXCL11, vCCL2, CXCL10, BAM22 or ADM at concentrations ranging from 6 pM to 1 μM. All binding experiments were performed in PBS containing 1% BSA and 0.1% NaN 3 (FACS buffer). Nonspecific chemokine binding was evaluated by the addition of 250fold excess of unlabeled CXCL12 and the signal obtained was used to define 0% specific binding. The signal obtained for CXCL12-AF647 in the absence of unlabeled chemokines was used to define 100% binding. Chemokine binding was quantified by mean fluorescence intensity on a BD FACS Fortessa cytometer (BD Biosciences).

The mAb 9C4 (3.3 μg/ml) was first incubated for 30 min at room temperature with peptides CCS, SSS (100 nM) or TPA (100 µM) derived from the N terminus of ACKR3. Peptide SSS scrbld (100 nM) was used as negative control. The antibody-peptide mix was then incubated for 60 min at 4 °C with U87 cells expressing wild-type ACKR3 (15 × 10 4 cells/well in a 96-well plate). The binding of 9C4 to ACKR3 was revealed with an allophycocyanin-conjugated goat anti-mouse IgG F(ab') 2 (Jackson ImmunoResearch) and quantified by mean fluorescence intensity on a BD FACS Fortessa cytometer (BD Biosciences).