Cp690550

The human HEK293T cell line was grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10 % fetal bovine serum (FBS; Atlanta Biologicals), 2 mM L-glutamine (Corning), and 1 % penicillin/streptomycin (Corning). Murine pro-B-cells, Ba/F3 (DSMZ), and human embryonic kidney cells, Hek293, were cultured in RPMI 1640 medium containing 10 % FBS, 2 mM L-glutamine, and 1 % penicillin/streptomycin. Ba/F3 cell culture media was supplemented with 1 ng/ml murine interleukin-3 (IL-3) (Peprotech). At 48 hours post-transduction cells were washed 3 times with, and resuspended in IL-3 free media. Cells were then analyzed by FACS using a Beckman Coulter Gallios^™ Flow Cytometer for GFP and propidium iodide (MP Biomedicals). Transformed Ba/F3 cells were seeded into 96 well plates at a density of 10⁴ cells per 100 μL. They were then treated at a range of 0 to 500 nM with CP-690,550 (Selleck Chemicals), 0 to 10 μM with NC1153 [3 (link)] or 0 to 10 μM with EP-009 [4 ] for 24 hours at 37 °C. Cellular viability was then determined by MTS assay (Promega) according to the manufacturer’s instructions in triplicate (n=3). MTS assay and FACS analysis was performed on Ba/F3 cells grown in IL-3 free media. Cell counting was performed by using a hemocytometer (Hausser Scientific) and trypan blue (MP Biomedicals).