Cd123 buv395

Mononuclear cells from the peripheral blood of 36 patients with AML indicated at leukapheresis due to hyperleukocytosis at diagnosis were obtained by separation of leukapheretic products on Histopaque 1077 (Sigma, Prague, Czech Republic). The cells were resuspended in RPMI 1640 medium with 10% fetal calf serum and with antibiotics (100 U/mL penicillin, 100 µg/mL streptomycin), and aliquots were used for RNA isolation and for analysis of surface markers by flow cytometry. Additional analyses of PD-L1 transcripts were performed from cDNA samples, which had been taken for routine clinical analyses and cryopreserved. A written informed consent with the use of biological material for research purposes was obtained from all patients. The study was approved by the Ethics Committee of the Institute of Hematology and Blood Transfusion of the Czech Republic as a part of the research project 16-30268A (June 2015).
Antibodies used were the following: CD45-V450 (#560367), CD4-BUV395 (#564724), CD8-BUV395 (#563795), CD19-BUV737 (#564303), CD123-BUV395 (#564195), CD371(CLL-1)-BB515 (#565926), CD135(FLT3)-AlexaFluor647 (#563494), and CD34-BV786 (#743534) were from BD Biosciences (Prague, Czech Republic); CD38-PE (#1P-366-T100) from Exbio (Prague, Czech Republic); PD-1-APC (#17-2799-42) and PD-L1-PE-Cy7 (#25-5983-42) from Affymetrix, Inc. (San Diego, CA, USA).

Absolute counts of peripheral blood leukocytes were determined for all study participants using BD Trucount tubes, six-color TBNK Reagent and, in addition, CD123 BUV395 (BD Biosciences), CD15 PB, CD193 BV605, and HLA-DR BV785 (Biolegend), and CD14 PE-Cy5 (eBioscience) according to the manufacturer’s instructions. Samples were fixed in 1× BD fluorescence-activated cell sorter (FACS) lysing solution (BD Biosciences) for 2 h and acquired on a BD FACSymphony A5 instrument, equipped with ultraviolet (355nm), violet (405 nm), blue (488 nm), yellow/green (561 nm), and red (637 nm) lasers. For absolute cell count calculations, the number of events for populations of interest was divided by the number of bead events and multiplied by the BD Trucount bead count.

In order to determine the absolute leukocyte numbers, trucount staining was performed. Briefly, 50 µL EDTA blood and 20 µL of antibody mix (6-color TBNK Reagent and CD123 BUV395 from BD, CD15 PB, CD193 BV605 and HLA-DR BV785 from Biolegend, and CD14 PE-Cy5 from eBioscience) were added in BD trucount tubes and incubated for 15 min at room temperature in the dark. The samples were then fixed using 430 µL 1× BD FACS lysing solution (BD Biosciences).