Facsdiva 6.1 cellquest software

Mononuclear cells were isolated by density gradient centrifugation at 400 g and 20°C for 30 min, diluted in 90% fetal bovine serum (FBS) plus 10% dimethyl sulfoxide (DMSO) and stored in liquid nitrogen. CD34⁺ cells from BM samples of three patients randomly selected in our cohort, were magnetically separated using MACS CD34⁺ microbead kit (Miltenyi Biotech GmbH). In particular, the molecular analysis of these patients revealed a FLT3-ITD with normal karyotype and two MLL rearrangements [t(9;11) and t(10;11)]. The identity of CD34 cells was validated by flow cytometry using FACSCantoII equipped with FACSDiva 6.1 CellQuest software (Becton, Dickinson and Company) using 20 µl of CD34 PerCP antibody (cat. no 340666; BD Biosciences) with an incubation of 30 min at 4°C.

Following treatment with 250 nM BRD4 and 10 µM curcumin, cells were washed twice with ice cold PBS and stained for 15 min at room temperature in calcium-binding buffer with PE-conjugated Annexin V (AnnV) and 7-Aminoactinomycin D (7-AAD) using the AnnV apoptosis detection kit (BD Pharmingen; BD Biosciences) according to the manufacturer's recommendations. Samples were analysed within 1 h by a fluorescence-activated cell sorting using a FACSCantoII equipped with FACSDiva 6.1 CellQuest software (Becton, Dickinson and Company).