CXCR4 Receptor Internalization Assay

CXCR4 receptor internalization was performed as previously described^{54 (link)}. Briefly, ALL cells were maintained in RPMI medium as described above. They were then stimulated with or without medium supplemented with 125 nM Dex or 100 nM SDF-1α, and kept either at 4 °C (for T = 0) or incubated at 37 °C for the times indicated in the figures. All subsequent steps were carried out at 4 °C. Cells were washed once in staining FACS buffer (PBS, 0.5% BSA, 0.05% NaN3, and 5% FBS) and incubated in the presence of anti-human CXCR4-APC (5:200 dilution, clone 12G5, eBioscience, France, cat. no. 17-9999-42) or isotype control APC-Mousse IgG 1k (1:200 dilution, clone MOPC-21, Biolegend, cat. no. 400119) / isotype control APC-Mousse IgG 1k (1:200 dilution, clone MOPC-21, BD Biosciences, cat. no. 554681) for 30 min. After two washes, signals were acquired on flow cytometer. Results are given as percentage of MFI controls, 100% corresponding to unstimulated (medium alone) cells processed in parallel. In some cases, Ca²⁺ signaling was also measured under the same conditions.