3.4. CXCR4 Calcium Mobilization Assay

The calcium mobilization assay has been described in detail previously [31]. U87.CD4.CXCR4 cells (2 × 10⁴ cells per well in DMEM/10% FBS/0.01 M HEPES) were seeded in gelatin-coated (Sigma-Aldrich; 0.1% gelatin in DPBS) black-walled 96-well plates and incubated overnight at 37 °C and 5% CO₂. The next day, cells were loaded with the fluorescent calcium indicator Fluo-2 acetoxymethyl (AM) ester (4 μM; Abcam) and incubated at room temperature in the dark for 45 min. Then, cells were incubated with various concentrations of the compounds for 10 min prior to the addition of 6.25 nM CXCL12 (in assay buffer). Fluctuations in intracellular calcium levels were measured in real time by the FLIPR Tetra^® (Molecular Devices, Sunnyvale, CA, USA) in all 96 wells simultaneously. The response over baseline (after CXCL12 addition) was calculated with the ScreenWorks 4.0^® software (Molecular Devices, Version 4.0, www.moleculardevices.com, accessed on 10 January 2015) by dividing the obtained relative light units (RLUs) through the base line measured just before CXCL12 addition. From this the IC₅₀ value for each compound was determined taking into account the negative (i.e., untreated cells without CXCL12 stimulation) and positive (i.e., untreated cells with CXCL12 addition) control samples.