The CD4+ Dendritic Cell Isolation Kit for mouse from Miltenyi Biotec allows for the isolation of a pure fraction of murine CD4+ dendritic cells from spleen tissue using a combination of negative and positive selection methods. CD4+ dendritic cells are of interest because they are the major subclass of dendritic cells in spleen tissue, and they can be used in antigen studies or in co-cultures with T cells to provide activation or polarization.
Prior to sorting, mouse spleens are subjected to digestion with Collagenase D (not provided in kit). This is different from the other cell isolation kits from Miltenyi (such as the T and B cell kits) which simply require teasing apart the spleen to release cells. The digestion procedure takes roughly one hour for the injection, disruption, incubation, and wash steps, and it is the longest part of the overall isolation procedure. Once the cells have been counted, pelleted, and resuspended in MACS buffer (not provided in kits, but recipe is listed in the protocol), it is very important to keep them on ice. All reagents should be cold, and the incubation steps should be performed on ice. This prevents non-specific binding of the antibodies, thus improving the resulting amount of purified dendritic cells.
The first sorting step uses a cocktail of biotin-conjugated antibodies which bind to all T, B, NK cells and granulocytes. Following this incubation, magnetic Anti-Biotin Microbeads are incubated with the cell mixture and become attached to only those cells which have the primary antibody bound. To sort the cells, MS and LS Columns or an autoMACS Separator from Miltenyi Biotech are needed. Both of these methods bind the magnetic cell fraction, allowing the non-labeled dendritic cell fraction to flow through. Then the magnetic field is removed and the positively labeled, magnetic non-dendritic cell fraction is eluted.
The second step involves directly labeling CD4+ dendritic cells in the non-labeled dendritic cell fraction with magnetic CD4 Microbeads. This cell fraction is then washed, pelleted, and resuspended for sorting. In this step, the positive fraction contains the cells of interest.
For sorting, I use the autoMACS Separator, specifically using the “Depl025” program for the first sorting step where the “negative” fraction contains the dendritic cells. Then the “Posseld2” program is used to sort out the positively labeled CD4+ dendritic cells.
The labeling steps are quick, with only 10 minutes for the antibody incubation and 15 minutes for each bead binding. There is no wash step between the antibody and bead labelings, which also reduces the procedure time. A wash step is needed, however, after the CD4+ Microbead incubation, but this only takes 10 minutes. This kit supplies enough antibody and microbeads for the sorting of 2x109leukocytes, which is the equivalent of roughly 20 mouse spleens.
Overall, the kit is easy to use and very effective. Because of the additional steps of collagenase treatment and CD4+ Microbead incubation, this kit takes longer than some other Miltenyi sorting kits. To check for purity, fluorescence activated cell sorting can be done using a fraction of the isolated CD4+ dendritic cells labeled with a fluorescent anti-CD11 antibody (dendritic cell marker), as well as with an anti-CD4 antibody with a different fluorochrome. I have done this with a few of my isolations and extremely high purity (>99%) is present in the isolated fractions using these kits.