Optibuild

Changes in the expression of various molecules in the compound-treated and control (DMSO-treated) cell lines were determined by flow cytometry, as described [12 (link)]. In brief, leukemic cells (10⁶) were first incubated with human Fc receptor binding inhibitor or CD16/CD32 blocking antibody (eBioscience, San Diego, CA, USA) for 20 min. Cells were then stained with the following primary antibodies for 1 h on ice. Primary antibodies were: Allophycocyanin-conjugated anti-mouse CD71 and Phycoerythrin-conjugated anti–mouse or anti-human CD41, CD61, Ter119, CD235a (eBioscience). After washing the cells with Phosphate Buffered Saline (PBS), a minimum of 10000 events were collected using the FACSCalibur flow cytometer (BD Biosciences, Beijing, China) and analyzed using CellQuest Pro software (BD Biosciences).
Antibodies for Flow cytometer analysis were obtained as follows; CD16 (#560248), CD41 (#555468), CD61 (#555753), Ter119 (#557915), CD235a (#555569), CD71 (#555534) and Annexin V (#550475) from Becton Dickinson (BD) (BD, Salt Lake City, Utah, USA), CD32 (#740399) and PI (#745153) from BD Optibuild (BD Biosciences, San Jose, CA, USA).

About 1 × 10⁶ cells were harvested, resuspended in 300 µl of 5% BSA dissolved in PBS and blocked for 30 min at room temperature. Then, cells were repelleted and resuspended in 100 µl of primary antibody diluted in 1% BSA in PBS and let for 1 h at room temperature in the dark. At this point, cells were washed with 1 ml of 1% BSA in PBS, centrifuged and resuspended in 250 µl of cold PBS. Transduced cells were, then, fixed by the addition of 250 µl 2% formaldehyde in PBS and incubated for 20 min in ice. After a further spinning, cell pellets were resuspended in 500 µl of PBS and analyzed by the FACS Celesta flow cytometer (BD Biosciences). Data analysis was performed using the FlowJo software. The antibodies used for the flow cytometry analysis of the cell surface markers were: anti-human CD11b (740965, BD OptiBuild™, 1:100), anti-human CD14 (11-0149-42, Thermo Fisher Scientific, 1:100), and anti-human CD117 known also as c-KIT (12-1178-42, Thermo Fisher Scientific, 1:100). Flow cytometry analysis of MLL- translocated primary patient cells are carried out as described in [12 ] and the antibodies used were: anti-human CD11b (12-0118-42, Thermo Fisher Scientific, 1:200), anti-human CD86 (46-0869-42, Thermo Fisher Scientific, 1:200), and anti-human CD14 (46-0149-42, Thermo Fisher Scientific, 1:200).