Miltenyi Biotech makes several kits that effectively isolate different immune cell types, such as T cells, B cells, or dendritic cells. These kits are especially useful for studies investigating the functions of specific types of immune cells. The CD4+ T Cell Isolation Kit and CD8+ T Cell Isolation Kit for mouse from Miltenyi Biotech allow for the isolation of a pure fraction of murine CD4+ T cells and CD8+ T cells, respectively, from spleen, lymph nodes, or blood by using negative selection.
In this procedure, cells are exposed to a cocktail of biotin-conjugated primary antibodies which bind to all non-T cells, such as B cells, NK cells, dendritic cells, macrophages, granulocytes, and erythroid cells. The CD4+ T cell kit also contains an antibody against CD8a, while the CD8+ T cell kit has an anti-CD4 antibody, allowing for removal of all cell types other than CD4+ or CD8+ cells, respectively. After antibody binding, magnetic beads (with bound anti-biotin antibody) are incubated with the cell mixture and become attached to only those cells with primary antibodies bound. Cells are then washed, pelleted, and resuspended for sorting. To sort the cells, either MS and LS Columns or an autoMACS™ Separator from Miltenyi Biotech is needed. Both of these methods bind the magnetic cell fraction, allowing the pure non-labeled T cell fraction to flow through. Then the magnetic field is removed and the positively labeled, magnetic non-T cell fraction is eluted.
I use these kits primarily on mouse spleen cells, and have also used them on mouse blood cells. For spleen cells, I tease apart the spleen in sterile PBS and lyse erythrocytes using RBC lysis buffer (from eBiosciences). For blood cells, I perform the erythrocyte lysis twice to ensure full removal of red blood cells. The protocols do not mention this step, however, I include this lysis to ensure that excess red blood cells won’t bind the majority of the primary antibodies. Following the lysis step, cells are washed in PBS and counted: I use the trypan blue exclusion method with a hemocytometer. I usually get around 108 cells from one mouse spleen, and just under 107 cells from 1 ml of blood. Both the CD4+ and CD8+ T cell Isolation Kits contain enough reagents for 109cells, or roughly 10 spleen isolations.
Once the cells have been counted, pelleted, and resuspended in MACS™ buffer (not provided in kits, but the recipe is listed in the protocol), it is very important to keep them on ice. All reagents should be cold, and the antibody incubation step should be performed at 4°C. This prevents non-specific binding of the antibodies to T cells, thus improving the resulting amount of purified T cells. The labeling steps are quick, with only 10 minutes for the antibody incubation and 15 minutes for the bead binding. There is no wash step between the antibody and bead labelings, which also reduces the procedure time. After the bead labeling, cells are washed and then resuspended for sorting. I usually resuspend my cells in at least 1ml of MACS™ buffer (twice as much as the protocol recommends) because this allows enough volume for adequate resuspension of the cells.
For sorting, I use the autoMACS™ Separator, which is an automated system that takes up the cell suspension, applies it to a column for binding, and elutes the non-labeled CD4+ or CD8+ T cell fraction into one tube and the labeled non-T cell fraction into another. At this point, cells are counted again and can be spun down and resuspended in the appropriate cell media for culturing or experimental use. I use these cells for long-term culturing, T cell activation studies, or T cell signaling studies. So far, I have had very good results using these cell isolation kits with my experimental procedures.
Overall, the kits are very easy to use, very quick (about 1 hour, going from total spleen cells to isolated CD4+ or CD8+ T cells) and very effective. To check for purity, fluorescence activated cell sorting can be done using a fraction of the isolated CD4+ or CD8+ T cells labeled with a fluorescent anti-CD4 or anti-CD8a antibody, respectively, as well as an anti-CD3 epsilon antibody with a different fluorochrome. The anti-CD3 epsilon antibody will label all T cells to check the purity of the T cell fraction, and the anti-CD4 and anti-CD8 antibodies will label each sub-population. I have done this with a few of my isolations and extremely high purity (>99%) is obtained in the isolated fractions using these kits. I fully intend on using these and other immune cell isolation kits from Miltenyi Biotech for my upcoming studies.