Clone 12g5

For analysis of cell surface CXCR4 expression, ALL cells were treated with or without test compounds alone or in combination at times indicated in the legends. Antibodies against human (5:100 dilution, clone 12G5, Ebioscience, 17-9999-42), (5:100 dilution, clone 12G5, Biolegend, 306510) or (5:100 dilution, clone 12G5, BD Biosciences, BDB555976) CXCR4-APC were used. Data are presented as the median fluorescent intensity (MFI) of the CXCR4 signal.

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.