The Spectacular Thriller Of Transducin
Mice were closely monitored for MPN development and harvested at moribund. For F-14 treatment in a primary transplant model, irradiated BoyJ mice were transplanted with 2.0?�� 106 GFP-KITD814V cells and 0.1?�� 106 supporting BM cells. Three weeks posttransplant mice were treated with 10?mg/kg body weight F-14 for 5?days a week for 6?weeks. All mice were harvested, and peripheral blood (PB) counts were monitored OTX015 datasheet using a Hemavet 950 (Drew Scientific). PB, BM, and spleens were analyzed for GFP expression. BM, spleen, and lungs were also fixed in 10% buffered formalin to perform histopathologic analysis by hematoxylin and eosin staining. Eight- to ten-week-old C57BL/6, PAK1?/?, or FAK?/? mice were injected with 5-flurouracil (150?mg/kg body weight), and after 5?days, the mice were killed and BM was harvested. Cells were prestimulated overnight in Iscove modified Dulbecco medium supplemented with 20% fetal bovine serum with mouse SCF (100?ng/ml), mouse TPO (100?ng/ml), this website mouse Flt3 (50?ng/ml), and mouse IL-6 (10?ng/ml). Cells were plated on Retronectin-coated plates (Takara), and retroviral transduction was performed using viral supernatants as described before ( Mali et?al., 2011). GFP+ve cells were sorted using FACS Aria (BD Biosciences). We injected 1?�� 106 GFP-positive cells along with 0.1?�� 106 supporting splenocytes into the tail veins of lethally irradiated (11 Gy) female C57BL/6 recipient mice. Peripheral blood counts were monitored on a regular interval after transplantation on a Hemavet 950 (Drew Transducin Scientific) and by fluorescence-activated cell sorting analysis for GFP expression. All moribund mice were then euthanized, and peripheral blood, BM, and spleen cells were analyzed for GFP expression. At day 120, all remaining mice were euthanized and analyzed. For secondary transplant, leukemic WT mice and time-point-matched PAK1?/? or FAK?/? mice, both bearing KitD814V-expressing cells, were harvested and 1?�� 106 cells were injected into the tail veins of lethally irradiated (11 Gy) female C57BL6 recipients. All graphical data were evaluated by paired Student��s t test (two-tailed), and results were considered significantly different with p value?
