By Shaoguang Li, Haojian Zhang
This quantity highlights the molecular and mobile tools utilized in studying Chronic Myeloid Leukimia (CML) pathogenesis and stem phone biology. Written within the hugely winning Methods in Molecular Biology series structure, chapters contain introductions to their respective issues, lists of the required fabrics and reagents, step by step, with ease reproducible laboratory protocols, and tips about troubleshooting and warding off identified pitfalls.
Authoritative and state-of-the-art, Chronic Myeloid Leukemia: equipment and Protocols goals to make sure winning ends up in the additional examine of this important field.
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Additional info for Chronic Myeloid Leukemia: Methods and Protocols
7. Spin cells at 500 Â g for 5 min again, and remove the supernatant. 8. Resuspend the cell pellet in fluorescence-activated cell sorting (FACS) buffer, and adjust cell concentration to 1 Â 107 cells/mL. 9. Stain cells with fluorescence-labeled myeloid cell marker (Gr-1): add phycoerythrin (PE) or allophycocyanin (APC)-labeled Gr-1 antibody to 50 μL of cells. Mix well, and stain the cells at 4 C for 15–30 min. 10. Add 1 mL of PBS buffer to the cells, and wash cells once by spinning at 500 Â g, 4 C for 5 min.
3. Remove the NIH3T3 cell medium, and add premixed virus supernatant for infection. After 3 h culture, remove the virus supernatant, and change to 10 mL fresh medium. 4. After 48 h postinfection, digest cells with trypsin, and collect cells into 1 mL flow cytometry buffer. Take 300 μL cell suspension to run flow cytometry analysis for percentage of GFPþ cells. Normally, the good retroviral supernatant means the % GFP can reach to 90–95 % at the 1:2 dilution, 75–85 % at 1:8 dilution, and 60–70 % at 1:16 dilution.
7. Incubate cells in incubator at 37 C, 5 % CO2 for 3 h. Note: If the cells accumulate in the corner, gently shake or blow the clot to make it loose. 8. After 3 h incubation, gently remove supernatant with pipette to new tubes and centrifuge at 300 Â g for 10 min to return the removed cells; continually culture the cells with 4 ml fresh stimulation medium overnight. 2 ml, 100Â penicillin/strep, 40 μl, 100Â L-glutamine 40 μl, Ciproxin 4 μg, IL-3 4 μg, Il-6 4 μg, and SCF 4 μg. Note: You could make the culture medium half hour before changing medium.