B Cell Isolation Kit From Miltenyi Biotec

The B Cell Isolation Kit for mouse from Miltenyi Biotech allows for the isolation of a pure fraction of murine B cells from spleen, lymph nodes, or blood by using negative selection. Using the kit, cells are exposed to a cocktail of biotin-conjugated antibodies which bind to all non-B cells, such as T cells, NK cells, dendritic cells, macrophages, granulocytes, and erythroid cells. Following this incubation, Anti-Biotin Microbeads, beads which are magnetic and have anti-biotin antibody attached, are incubated with the cell mixture and become attached to only those cells which have the primary antibody bound. Cells are then washed, pelleted, and resuspended for sorting.

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 pure non-labeled B cell fraction to flow through. Then the magnetic field is removed and the positively labeled, magnetic non-B cell fraction is eluted.

I use this kit to isolate cells from mouse spleens. To do this, I tease apart the spleen in sterile PBS and lyse erythrocytes using RBC lysis buffer (from eBiosciences). The protocol does 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, counted, and kept on ice prior to the antibody binding. I usually get around 10⁸cells from one mouse spleen, which means the kit provides enough reagents for roughly 10 spleen isolations.

Once the cells have been counted, pelleted, and resuspended in MACS buffer (not provided in kit, but the recipe is listed in the protocol), it is very important to keep them on ice. All steps of the B cell Isolation Kit should be performed on ice to prevent non-specific binding of the antibodies to B cells, thus decreasing the yield of purified B cells. The labeling steps are quick, with only 10 minutes for the antibody incubation and 15 minutes for the bead binding. After the bead labeling, cells are washed and then resuspended for sorting. I usually resuspend my cells in at least 1 ml 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 B cell fraction into one tube and the labeled non-B 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. After B cell sorting, I usually get around 5x10⁷B cells from one spleen. So far, I have had very good results using this cell isolation kit combined with my experimental procedures.

Overall, the kit is very easy to use, very quick (about 1 hour to go from total spleen cells to isolated B cells) and very effective. To check for purity, a fraction of the isolated B cells can be labeled with a fluorescent anti-CD19 or anti-CD45R antibody for fluorescence activated cell sorting. I have done this with some of my isolations and extremely high purity (>99%) is obtained in the isolated fraction using this kit.