Thymic stromal lymphopoietin tslp

To identify Jak2/Stat5, ABL, and Ras pathway alterations in ALL patient samples, fresh cells from bone marrow were stimulated with 200 ng/ml thymic stromal lymphopoietin (TSLP) (PeproTech, Cranbury, NJ, USA) for 30 min at 37 °C, and stained with the following cell surface monoclonal antibodies: CD45 Amcyan, CD34 PECy7, CD19 PerCP, and CD10 PE (Becton Dickinson, USA) [32]. Then, the cells were fixed with BD Phosflow Fix Buffer I, permeabilized with BD Phosflow Perm Buffer III (Becton Dickinson), and incubated with monoclonal antibodies to identify phosphorylated targets of each pathway: Stat5 (pY694) Pacific Blue to identify Jak2, CrkL (pY207) AF488 for ABL, and Erk 1/2 (pT202/pY204) AF488 for Ras. In cases with an adequate number of cells, the abnormal pathway activity was selectively inhibited in vitro for 30 min with ruxolitinib (5 nm) (Selleckchem, Houston, TX, USA) to inhibit Jak2/Stat5 pathway, and with imatinib and/or dasatinib (5 μm) (Selleckchem) to prevent ABL abnormal activation. The antibody concentration and conditions for each assay were performed based on the manufacturer's recommendations. Flow cytometry was performed using a BD FACSVerse Cell Analyzer System and data were analyzed by FlowJo vX software.