The probes and primers for B 2 microglobulin and MCL 1 were purchased from Applied Biosystems. MTT assays and synergy calculations Cytotoxicity assays were performed with the MTT 2,5 diphenyl tetrasodium bromide reagent. Five-hundred thousand CLL cells resuspended in AIM V medium were Dovitinib PDGFR inhibitor plated per well in flat bottomed 96 well plates and confronted with serial doubling concentrations of drug for 72 hours. For the last 6 hours, 0. 5 mg/ml MTT was added before also adding 10% SDS with 0. 01 M HCl. After incubation over night at 37 C, absorbance was measured at the wavelengths of 650 nm and 570 nm. The difference between the absorbance measurements at test and reference wavelengths was used to match a dose response curve, and the required drug concentration to kill 500-watt of the cells, the IC50, was determined by non-linear regression using Prism 4. 0. Vehicle treated cells served as controls. Synergy between materials was assessed with CalcuSyn software according to the process described by Chou and Talalay. Mathematical investigation Gene expression Unpaired and matched T-tests were used to determine differences in means of two teams for cell viability and CD44 expression. A G value 0. 05 was considered important. CD44 expression varies between prognostically unique CLL sub-types High expression of CD44 on CLL cells has been associated with adverse clinical features. But, the connection between expression and the more recently defined prognostic subtypes of CLL and in particular with IgVH mutational status or ZAP70 expression has not been identified. Using movement cytometry, we quantified CD44 expression in CLL cells and in T lymphocytes obtained from healthier donors. Surface CD44 was discovered on normal T cells along with on all CLL cells. The amount of CD44 expression linked with IgVH mutational status and was highly variable among different CLL samples. To evaluate the expression of CD44 we determined the ratio between the mean Dasatinib structure fluorescent intensity of CD44 staining divided by the MFI of the corresponding isotype staining. The expression of CD44 was somewhat higher in U CLL cells than in M CLL cells or in normal B cells. On the other hand, MCLL cells had lower CD44 expression than normal B cells. CD44 triggers homotypic region and protects CLL cells from spontaneous apoptosis To analyze the effect of CD44 signaling on CLL cells, we first stimulated PBMCs from CLL patients with a monoclonal antibody that binds to the extra-cellular domain of CD44. CD44 engagement triggered homotypic aggregation of the CLL cells, which is a common aftereffect of various exogenous stimuli that activate cells or regulate cell adhesion. CLL cells aggregated within a few minutes and clustered into clumps containing many cells. These sections were seen as a strong cell-cell interactions and were difficult to dissociate.