Screenworks 4

High-throughput FLIPR assays were conducted using 384-well BioCoat (Corning) plates. Cells were washed briefly in assay buffer (HyClone™ HEPES-buffered saline [GE Life Sciences] comprising 149 mM NaCl, 4 mM KCl, 10 mM HEPES, and 5 mM glucose at pH 7.4 and 300 mOsm osmolality, supplemented with 2 mM CaCl₂ and 1 mM MgCl₂) prior to Calcium5 dye (Molecular Devices) loading for 1 h at RT. Following removal of excess dye, plates were placed in the FLIPR Tetra (Molecular Devices) chamber, and fluorescent Ca²⁺ signal was captured using ScreenWorks 4.0™ software (Molecular Devices).
To obtain neuronal α7 mediated FLIPR responses, we used 5 μM PNU-12059615—a selective drug that attenuates receptor desensitization. Tetrodotoxin, or TTX (500 nM) was included in the assay buffer to inhibit spontaneous action potentials.

The calcium (Ca²⁺) mobilization assay was described in detail before [24 (link)]. Briefly, U87.CD4.CXCR4 or U87.CD4.CCR5 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 Ca²⁺ indicator Fluo-2 acetoxymethyl (AM) ester (4 μM final concentration; Abcam) and incubated at RT in the dark for 45 min. Then, cells were washed with assay buffer (see above) and subsequently incubated with different concentrations of compound for ~ 10 min prior to addition of 6.25 nM CXCL12 (in case of CXCR4) or 6.4 nM CCL5 (in case of CCR5). Fluctuations in intracellular Ca²⁺ levels were measured in real time by the FLIPR Tetra^® device (Molecular Devices) in all 96 wells simultaneously. The response (Relative Light Units, RLUs) over baseline was calculated (ScreenWorks 4.0^® software, Molecular Devices) by dividing the RLUs by a base line value measured just before addition of CXCL12 or CCL5, respectively. Inhibition of the Ca²⁺ response was determined taking into account negative (i.e., untreated cells without stimulation) and positive (i.e., untreated cells stimulated with either CXCL12 or CCL5) control samples.

Apparent binding kinetics (K_D, k_a, k_d) were derived after fitting the experimental data to the 1:1 Langmuir binding model using the Biacore T200 Evaluation Software 3.1. Data obtained from this software program were transferred to Graphpad Prism 8 for production of the sensorgrams. The sensorgrams start at 0 RU when the analyte is injected, the 400–600 RU immobilization response is automatically corrected to zero. Means and standard deviations were calculated using Excel. Simple linear regression of the correlation data was calculated using Graphad. Flow cytometry data were analyzed with the FlowJo^® Software (Ashland, OR, USA). The affinity (K_D) derived from the flow cytometry data represents the Nb-Fc concentration needed to bind 50% of the CXCR4-expressing cells at equilibrium. The calcium mobilization data were analyzed with the ScreenWorks 4.0 software (Molecular Devices, Sunnyvale, CA, USA). Relative light units (RLUs) were corrected by subtracting the RLU measured at a specific time point just before CXCL12 addition from the RLUs measured on all other time points. Next, the difference between the maximum and minimum corrected RLUs was calculated.