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The labeled T cells (1.25 �� 105/mL) were cultured alone or with leukemia cells (2.5 �� 105/mL) in the presence www.selleckchem.com/products/ve-822.html or absence of functional stimulatory or blocking antibodies (as described in the co-culture assays). Following 96-h incubation, cells were harvested and analyzed by flow cytometry. The supernatants were collected directly from HL-60 and iHL-60 cells; or from T cells, HL-60, or iHL-60 cells back-sorted from the co-cultures following overnight incubation (2.5 �� 105 cells/mL). Equal volumes of supernatants obtained from at least three independent experiments were pooled. ELISAs were performed with Human Th1/Th2/Th17 Cytokines Multi-Analyte ELISArray Kit and Human Inflammatory Cytokines Multi-Analyte ELISArray Kit (SABiosciences, Qiagen, Valencia, CA, USA) according to the manufacturer's instructions. Data presented in this study are representative of at least three independent experiments. All values are expressed as arithmetic mean �� SD. Statistical difference between experimental groups was determined using Student's paired or unpaired t-test where appropriate. Differences were regarded as statistically significant when p �� 0.05. This study was funded by Hacettepe University Research Unit (Project No. 011 D04 104 001), and by Ankara Guven Hospital. G. Esendagli was supported by the ��Dr. Aysun-Ahmet Kucukel Young Researcher Scientific Support Award.�� We thank Hande Canpinar, PhD and Parisa Sarmadi, MSc for additional support in the conduct of experiments. The authors declare no financial or commercial conflict of interest. As a service to our authors and readers, this journal provides https://www.selleckchem.com/products/3-methyladenine.html supporting information supplied by the authors. Such materials are peer reviewed and may be re-organized for online delivery, but are not copy-edited Ibrutinib or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors. Supporting Information Fig. S1. Gating strategy used in flow cytometric analyses. Co-cultured lymphocytic and myelocytic cells were initially gated according to side scatter (SSC) and forward scatter (FSC) properties. The cells were re-gated according to staining with a myeloid marker (designated as fluorescence channel (FL1)). Then, the expression of several surface molecules on myeloid leukemia cells and/or T cells was analyzed on FL2 or FL3. Supporting Information Fig. S2. Immunophenotypic analysis of HL-60 and iHL- 60 cells was performed for the determination of FAB category. (A) Representative flow cytometry histograms and (B) quantification of the immunophenotypic markers used in AML diagnosis and classification are given. Overlay graphs are shown with specific mAb staining (filled histograms) and with isotype-matched control staining (open histograms). Data were obtained from five independent experiments and are shown as mean �� SD (Student's t-test, **P