By Stephen P. Robinson, Andrew J. Stagg
Stephen P Robinson, MD, PhD, and Andrew Stagg, PhD, have introduced jointly a variety of time-proven tools for learning those dendritic cells. a lot of those easily reproducible concepts care for the matter of acquiring adequate dendritic cells for research, no matter if by way of isolation from a large vareity of tissues, or from quite a few progenitor phone populations. different tools describe in step by step type the suggestions wide-spread for studying facets of dendritic cells, starting from cellphone migration to antigen uptake and T cellphone stimulation. additionally, a couple of innovations discover the sensible demanding situations occupied with utilizing dendritic cells in a medical atmosphere to improve novel immunotherapeueutics.
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Additional info for Dendritic Cell Protocols (Methods in Molecular Medicine)
However, by carefully following the procedure as described above, DC that are “near-physiological” can be obtained with good yields and high purity. 2. 1. Digestion of Spleens 1. 5 cm sterile Petri dish with 3 mL of enzyme cocktail. 2. Fill a sterile 5 mL syringe with 2–3 mL enzyme, and using a 23G needle inject the enzyme into several sites in the spleen. When inserting the needle into the spleen, use sterile forceps to hold the tissue in place and pierce the tissue with caution so as not to exit the other side.
In contrast to previously described DC isolation methods and with the exception of the enzymatic digestion step, this isolation method is performed entirely at 4°C thereby avoiding prolonged incubation steps at 37°C, which might alter the phenotypic and functional characteristics of DC (5). 055 g/mL. , 10 times less than the low-densitiy cell fractions used in previous protocols. The use of such VLDF makes the subsequent magnetic bead depletion more efficient and less expensive. 1% of total thymocytes (4), a minimum number of thymuses are needed in order to obtain a reasonable number of purified From: Methods in Molecular Medicine, vol.
4. Using a cell scraper gently press cells out of the spleen by working the scraper from the middle to the lower end of the spleen. 5. When capsule is clear turn the spleen round, make a hole at the other end of it and work the rest of the cells out. 6. When all cells have been removed, discard the empty capsule and transfer the cells to a conical tube. 7. Repeat the procedure with additional spleens, pooling the released cells. 8. Adjust to the appropriate volume (see below). 2. Enrichment of DC Using Metrizamide Gradients Following overnight culture, a single separation step on a metrizamide gradient enriches DC up to 100-fold.