PBMC samples were seeded (1 × 104 cells/well) in complete medium with or without the indicated concentrations of compounds. After 72 h incubation, cells were washed with PBS and stained with CD45RA PE (BD-Biosciences), CD62L APC (Miltenyi Biotec), CD3 FITC (Miltenyi Biotec) at room temperature for 20 min. Data collection was done on FACSVerse and dead cells were excluded from the analysis, based on the use of PI (Sigma-Aldrich).
For the evaluation of hENT2 protein expression, cell lines or AML primary cells were incubated with a SLC29A2 polyclonal antibody (Thermo fisher Scientific) using a dilution of 1:25 for 20 min at room temperature after fixation and permeabilization. The secondary antibody (Cell signaling Technology USA, Danvers, MA, USA) was used with a dilution of 1:200 for 20 min. Results were expressed as ΔGmean, i.e., the net mean fluorescence intensity (MFI) difference between the cells stained with primary and secondary antibodies and cells stained with the secondary antibody only, in order to eliminate the background positivity. All data were processed with Kaluza software version 1.2 (Beckman Coulter).
For the evaluation of hENT2 protein expression, cell lines or AML primary cells were incubated with a SLC29A2 polyclonal antibody (Thermo fisher Scientific) using a dilution of 1:25 for 20 min at room temperature after fixation and permeabilization. The secondary antibody (Cell signaling Technology USA, Danvers, MA, USA) was used with a dilution of 1:200 for 20 min. Results were expressed as ΔGmean, i.e., the net mean fluorescence intensity (MFI) difference between the cells stained with primary and secondary antibodies and cells stained with the secondary antibody only, in order to eliminate the background positivity. All data were processed with Kaluza software version 1.2 (Beckman Coulter).