By Shaoguang Li, Haojian Zhang
This quantity highlights the molecular and mobile equipment utilized in studying Chronic Myeloid Leukimia (CML) pathogenesis and stem mobilephone biology. Written within the hugely winning Methods in Molecular Biology series layout, chapters comprise introductions to their respective themes, lists of the required fabrics and reagents, step by step, comfortably reproducible laboratory protocols, and tips about troubleshooting and heading off recognized pitfalls.
Authoritative and state of the art, Chronic Myeloid Leukemia: tools and Protocols goals to make sure winning leads to the extra research of this important field.
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Additional info for Chronic Myeloid Leukemia: Methods and Protocols
9 Chr. 9q+ HSC Ph 22q- Chr. 22 BCR-ABL BCR ABL Fig. 1 Philadelphia chromosome and three different forms of the chimeric BCRABL oncogene. CML originates from an abnormal hematopoietic stem cell harboring Philadelphia chromosome. Due to chromosomal translocation, the ABL gene on chromosome 9 fuses the BCR gene on chromosome 22 to form the fusion oncogene BCR-ABL of progenitor cells. In blast crisis, the disease displays arrested hematopoietic differentiation and accumulation of immature blast cells resembling acute leukemia.
Elute BM cells from the femurs and tibias with 293T medium by 10 ml syringe with 27G1/2 needle into 50 ml tubes. 4. Suspend cells with 10 ml pipette up-down to dissociate cells, then filter cells with 100 μm cell filter. 5. 5 ml Eppendorf tube, put tube on ice for no less than 10 min, add 25 μl 1 % trypan blue, count cells with microscope, and calculate total cell number. 6. Spin down cells at 300 Â g for 10 min. 32 Yiguo Hu 7. Loose cell pellet by flicking the tube, and resuspend cells with 10 ml stimulation medium and transfer them into 10 cm cell culture dish.
05. 2. The condition of 293T cells is also important for generating higher-titer virus. In addition, the confluence of the cells should reach ~90 %. 3. Virus-producing 293T cells are easy to detach from culture dish, medium should be changed carefully by adding it to the culture dish very slowly. 4. To avoid repeated freeze–thaw cycle, we recommend to aliquot virus supernatant after collection. 5. When making 5-FU solution for priming recipient mice, incubation in a 37 C water bath followed by vortexing is recommended to help to fully dissolve 5-FU.
Chronic Myeloid Leukemia: Methods and Protocols by Shaoguang Li, Haojian Zhang