By Elodie Segura, Nobuyuki Onai
The 3rd version of this quantity is aimed toward supplying either novices and more matured researchers a decision of how to isolate and learn dendritic cells(DC). An introductory evaluate presents an outline of modern advances within the characterization of DC subsets in mouse and human. whereas extra chapters offer how to tradition human and mouse dendritic cells, protocols for the isolation of dendritic cells, the isolation of dendritic phone progenitors from mouse, and the purification of dendritic cells from human blood. Written within the hugely winning Methods in Molecular Biology series layout, chapters comprise introductions to their respective subject matters, lists of the mandatory fabrics and reagents, step by step, effortlessly reproducible laboratory protocols, and tips about troubleshooting and warding off recognized pitfalls.
Authoritative and cutting-edge, Dendritic mobile Protocols, 3rd version aims to make sure winning leads to the extra research of this very important field.
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12. The day after, transfer the vials to liquid nitrogen for longterm storage. 5 Revival of Frozen Expanded Hematopoietic Precursors 1. Set the water bath at 37 °C. 2. Transfer the vials to the water and thaw the cells rapidly until only a small piece of ice is left in the tube (see Note 9). 3. Transfer the cells to a 15 mL polypropylene tissue culture tube. 4. Dilute the cell suspension fivefold in cold IMDM, 5 % FCS, 20 U/mL DNase I. 5. Gently mix the cell suspension, on ice. 6. Centrifuge the cell at 450 × g for 5 min at low break.
9. 10 cm Petri dishes. 10. Puromycin. 11. Humidified incubator set at 37 °C with 5 % CO2. 1 Derivation of CD8+ DC Lines from Tumor-Bearing Mushi Mice 1. When mice start looking pale, measure their hematocrit (see Note 7). Bleed the mice. 2. Collect the blood in a microcapillary. 3. Seal the microcapillaries with Critoseal Capillary Tube Sealant. 4. Spin the microcapillaries (15,000 × g for 3 min) in a microhematocrit centrifuge with a hematocrit rotor. 5. Determine the hematocrit by placing the tube in a microhematocrit tube capillary reader.
12. If you plate multiple wells, ensure that you add the medium before distributing the cells and gently mix after distribution. Otherwise, the cells may adhere quickly at high local concentrations. 13. During this phase, fibroblasts may overgrow which will terminate the culture. To avoid this, we prepare many replicates and discard the ones in which fibroblasts overgrow. 14. It can take up to 6 months and up to ten passages to obtain easy growing functional DC lines. 15. It is important to keep track of the passage numbers to avoid selecting for lines that have lost some of their functions.
Dendritic Cell Protocols by Elodie Segura, Nobuyuki Onai