The Miltenyi Biotec MACS (Magnetic Cell Sorting) system enables the separation of different cell populations by their expression of different surface antigens. In conjunction with the use of specific antibodies, subsequent sorting and separation strategies can enrich target cells, allowing direct comparison of cellular properties, including DNA/RNA/protein extraction, micro-array and in vitro cell culture, making this a powerful system for cellular analysis.
The theory behind the Miltenyi Biotec MACS technique is quite straightforward. First, cells are labeled using a primary antibody, specific to the cellular antigen of interest. This antibody does not need to have specific fluorescent or biotinylation properties, although a current limitation of the MACS system is that the antibody must be a rat IgG class. Second, using Miltenyi Biotec’s “colloidal super-paramagnetic” microbeads (goat, anti-rat IgG), the cells are labeled once again. The cell suspension is loaded onto a Miltenyi wire-mesh separation column (available in a variety of sizes based on cell density) and placed into a strong magnetic field (large, free-standing magnets specific to column size), where unbound cells are eluted as the “negative” fraction. The positive, labeled fraction of cells can also be easily eluted by demagnetizing the column.
I have used the MACS system for separation of several populations of mouse bone marrow cells, including granulocytes, stem/progenitor cells, T-cells, B-cells and macrophages/monocytes. By combining the initial magnetic cell separation with further antibody staining (using fluorescently-conjugated antibodies), we obtained more discrete cell separation by flow cytometry. We also used the MACS system for obtaining large numbers of cells for RNA extraction and subsequent RT-PCR.
In practice, we found that the columns were fairly durable (could be used more times than manufacturer suggested) and gave very good separation of labeled/unlabelled cells. The columns also did not impact cell viability and cells could be used for subsequent culturing, assuming proper sterile techniques were practiced throughout the separation.
This system does have several drawbacks. The initial purchase cost of the magnet and sorting columns is prohibitive and on-going replacement of the magnetic microbeads is also reasonably expensive. The cell-surface antigenic profile of many putative target cell populations is still unknown and furthermore, the system is currently limited to use exclusively with primary antibodies produced in rats. Although the theory of MACS is relatively straightforward, the practical applications and experimental design must be carefully researched and optimized, thus MACS is not ideal for inexperienced users. Still, the results to be gained from using cell-separation experiments can often be fast, plentiful and most importantly, biologically relevant.
Seb Dworkin
Graduate Student
Peter MacCallum Cancer Centre