Facslysing solution

The contents of each well of the cell culture plate were transferred to a 5 mL polystyrene tube (12 × 75 mm) and centrifuged for 5 min (540× g). The supernatant was discarded, and the cell pellet was stained with the monoclonal antibodies (mAb) described in detail in Table 2. After a 10 min incubation period in the dark and at room temperature, cells were fixed via 10 min incubation with 1 mL of FACSLysing Solution (BD). Cells were centrifuged (540× g, 5 min) to discard the FACSLysing Solution and washed with 1 mL of Dulbecco’s phosphate-buffered saline (PBS, Corning, Manassa, VA, USA). Finally, the cell pellet was resuspended in 500 µL of PBS and immediately acquired in a FACSLyric flow cytometer (BD) using FACSuite acquisition software (v1.5.0.925, BD, San Jose, CA, USA).

PB immune cells were identified and characterized by flow cytometry, using a stain–lyse–wash procedure previously described [20 (link),21 (link),22 (link)]. In summary, 100 µL of PB was incubated with the monoclonal antibodies (mAbs) indicated in Table 4, in the presence of 50 µL of Brilliant stain buffer (Becton Dickinson Biosciences (BD), San Jose, CA, USA), for 30 min in the dark and at room temperature. Subsequently, erythrocytes were lysed using 2 mL of FACSLysing solution (BD) and a 10 min incubation period. After centrifugation at 540× g for 5 min, the FACSLysing solution was discarded, and the resulting cell pellet was washed with 2 mL of Dulbecco’s phosphate-buffered saline (DPBS; Corning, Manassa, VA, USA). Lastly, the cell pellet was resuspended in 500 µL of DPBS, and the sample was acquired in a FACSLyric^TM (BD) flow cytometer, using the FACSuite acquisition software (v1.5.0.925; BD).