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Dendritic Cell Protocols
Buy eBook. FAQ Policy. About this book It is now apparent that dendritic cells not only play important roles in the body's immune system through their complex interactions with T cells, B cells, and other cell types, but also possess distinct functional attributes that enable them to assume different roles in that system. Show all.
Show next xx. Read this book on SpringerLink. Add the same volume of Buffer 1, or at least 1 ml, and mix.
Place the tube in a magnet for 3 min and discard the supernatant. Remove the tube from the magnet and resuspend the washed Dynabeads in the same volume of Buffer 1 as the initial volume of Dynabeads step 2.
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Sample Preparation As the enriched DC population is intended for further isolation of subpopulations by flow sorting we advise running single cell suspensions of spleen or lymph node cells through a density gradient to remove platelets, cell debris and high density cells 2. This protocol is based on isolation from a maximum of 5 spleens. Flush the cell strainer with the initial buffer to wash out all cells. Incubate the tube for 15 min at room temperature RT with tilting and rotation.
Add 0. Mix and incubate for 5 min on a roller at RT with tilting and rotation. Perform further cell processing on ice with cold buffers.
Dendritic Cell Protocols | SpringerLink
Fill the tube with Buffer 1. Discard the supernatant and resuspend the cell pellet in 5 ml of Buffer 1. Transfer the cell suspension and layer carefully on top of 4 ml density gradient medium in a 15 ml tube. Carefully transfer the leucocyte layer to a new tube.
Enrichment of Mouse DCs This protocol is based on enrichment from 1 x 10 7 leucocytes. It is scalable from 1 x 10 7 -5 x 10 8 cells, see table 1. Wash the cells by adding 2 ml Buffer 1. Discard the supernatant. Resuspend the bead-bound cells by thorough pipetting or vortex for 5 secs. Add 1 ml Buffer 1.
Dendritic Cell Protocols
Place the tube in the magnet for 3 min. These migratory DCs migrate to secondary lymphoid organs upon microbial infection and infl ammation, leading to their activation and differentiation , and present encountered antigens to T cells. The in vitro life span can be slightly extended by adding growth and differentiation factors or by using Bcl-2 overexpression or cells defi cient for proapoptotic molecules [ 7 ]. Injection of Flt3L into mice, adoptive transfer of tumors secreting Flt3L, and more recently, generating transgenic mice with Flt3L overexpression allowed generation of different DC sub- sets in the spleen in about fold higher numbers than from nor- mal mice [ 8 , 9 ].
All these lines have been cultured for many passages and have lost many of the initially described functions typical of DCs.