Cd34 bv786

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).

Fibronectin fragment (120 kDa cell attachment region) was from Merck (F1904).
IPA-3 (#3622) and PIR3.5 (#4212) were purchased from Tocris Bioscience and dissolved in sterile dimethylsulfoxide (DMSO) to make 50 mM stock solutions. Working solutions were prepared by 10-fold dilution of the stock solution in 50 mM Tris, pH 8.0, immediately before use. FRAX597 (S7271) and dasatinib (S1021) were obtained from SelleckChem, stock solutions were made in DMSO at 10 mM or 200 µM concentration, respectively, and diluted in culture media immediately before use. In all experiments, the cell density was adjusted to 3 × 10⁵ cells/ml prior to treatment with inhibitors.
The antibodies used were the following: PAK1 (Cell Signaling, #2602), PAK1 (Abcam, ab223849), PAK2 (Abcam, #ab76293), PAK1 (phosphoS144)+PAK2 (phosphoS141)+PAK3 (phosphoS139) (Abcam, #ab40795), β-actin (Sigma-Aldrich, A5441), CD45-V450 (BD Biosciences, #560367), CD34-BV786 (BD Biosciences, #743534), Integrin β1-AlexaFluor647 (Abcam, ab193592), Integrin αVβ3-FITC (Abcam, ab93513). All PAK antibodies were previously characterized using siRNA [29 (link)].