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 profitable Methods in Molecular Biology series layout, chapters comprise introductions to their respective themes, lists of the mandatory fabrics and reagents, step by step, without difficulty reproducible laboratory protocols, and tips about troubleshooting and keeping off recognized pitfalls.
Authoritative and state-of-the-art, Chronic Myeloid Leukemia: equipment and Protocols goals to make sure profitable ends up in the extra learn of this very important field.
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Additional info for Chronic Myeloid Leukemia: Methods and Protocols
Change 4 mL fresh 293T medium to each dish before transfection. 3. Prepare transfection cocktail per 6-cm dish: In a 15 mL tube, add 10 μg MIG-BCR-ABL-GFP plasmid, 5 μg EcoPack plasmid, 62 μL 2 M CaCl2, and sterile water to 500 μL total volume. Briefly vortex. 4. Add 500 μL 2Â HBS to the tube drop by drop, and mix by vortexing for 10 s (see Note 3). 5. Add DNA/HBS solution onto 293T cells drop by drop, and gently rock the dishes forward and backward a few times to achieve even distribution of DNA/Ca3(PO4)2 particles, and then continue to culture.
Use 5–10 μl of each Ab solution for 1 Â 107 cells suspended in 1 ml PBS containing 5 % FBS. 3. Use a flow cytometry such as a FACS Aria III instrument (BD Biosciences) or a similar instrument to isolate the cell fraction that is BCR-ABL1-GFP positive, cKit positive, lineage (CD4, CD8, B220, Mac1, Gr-1, and Ter119) negative, and Sca-1 positive. These cells constitute the CML-MPP (KLS) cell population, which includes CML LSCs. 2 Isolation of Murine LT-CML LSCs from the Double tg-CML Mouse Model 1.
7. After another 48 h culture, collect cells and resuspend cells with 1Â PBS; aliquot ~1 Â 105 cells into FACS tubes for flow cytometry analysis. Virus titer is determined with the percentage of GFP-expressing cells. If GFP% reaches to 90–95 % at 1:2 dilution, 75–85 % at 1:8 dilution, and 60–70 % at 1:16 dilution, the virus titer is high enough for BM cell transduction. 2 Collecting BM Cells from Primed Donor Mice 1. Dissolving 5-FU in PBS at 37 C at 10 mg/ml in dark. 2. 2 ml per 10 g weight).