Immunomagnetic cell separation is based on antibodies coupled to magnetic beads. During incubation with a cell suspension, the antibody/bead complex binds to cells expressing the corresponding epitope. When the cell suspension is placed into a magnetic field, magnetically labeled cells are retained, while unlabeled cells can be removed. To recover the labeled cells, the sample is removed from the magnetic field.
Different systems are available for immunomagnetic isolation of cells. Miltenyi Biotec offers i) a column-based technology that relies on nano-sized superparamagnetic MACS® MicroBeads and ii) a column-free technology using micro-sized superparamagnetic MACSxpress® Beads. Features of these technologies and the benefits over other technologies are explained in the following chapters.
|Positive selection of a target cell type|
|Depletion of an unwanted cell type|
|Untouched isolation of a target cell type|
|Depletion followed by positive selection|
|Two consecutive positive selections based on MultiSort MicroBeads and conventional MicroBeads|
|Two consecutive positive selections or positive selection followed by depletion based on REAlease® Technology|
Positive selection means that a particular target cell type is magnetically labeled. During separation, the column is placed in the magnetic field of the MACS Separator. Magnetically labeled cells are retained within the column, whereas unlabeled cells flow through. After a washing step, the column is removed from the magnetic field, and the target cells are eluted from the column. Specific MACS MicroBeads are available for the positive selection of numerous cell types and cell subsets.
Positive selection can be performed by direct or indirect magnetic labeling. In the case of direct magnetic labeling, the cell type–specific MicroBeads directly bind to antigens on the cell surface. For an indirect labeling approach, primary antibodies that bind to cell surface antigens and MicroBeads that bind to the primary antibodies are added to the cells separately in a two-step procedure.
For details about different options to label cells, see chapter Labeling strategies below.
To isolate a particular target cell type in an unlabeled, i.e., untouched form, non-target cells are magnetically labeled and depleted. During separation, the unlabeled target cell type is collected in the flow-through fraction. The mixture of magnetically labeled non-target cells is retained within the column. Optionally, the magnetically labeled cells can be eluted after removal of the column from the magnetic field (not shown).MACS Cell Isolation Kits for untouched isolation contain a cocktail of titrated biotinylated antibodies and MACS Anti-Biotin MicroBeads for indirect magnetic labeling. This approach is especially useful when binding of antibodies to the target cells is not desired.
This approach is useful if an important marker for the target cells is also expressed on a fraction of undesired cells. To enable positive selection of the target cells based on this marker, the fraction of undesired cells needs to be depleted first. To this end, the undesired cells are magnetically labeled via antigens distinct from that common marker. During separation, the labeled cells are retained in the column. The flow-through fraction contains the target cells. These cells can then be labeled with MACS MicroBeads for that marker, and the target cells are isolated by positive selection.
Sophisticated MACS Cell Isolation Kits based on this strategy are available for the fast and convenient isolation of specific cell subsets.
Direct labeling with MACS MicroBeads is the fastest way of magnetic labeling. MACS MicroBeads specifically bind to antigens on the cell surface. Only one incubation step is required. Direct magnetic labeling requires a minimal number of washing steps and therefore minimizes cell loss.Highly specific cell separation reagents for direct labeling of numerous cell types with MACS MicroBeads are available for human, mouse, rat, and non-human primate cells.
Indirect magnetic labeling using primary antibodies and MicroBeads is based on a two-step procedure. First, the cells are labeled with a primary antibody directed against a cell surface marker. Subsequently, the cells are magnetically labeled with a MACS MicroBead, which either binds directly to the primary antibody or to a molecule that is conjugated to the primary antibody. Conjugated molecules include biotin and fluorochromes. Accordingly, magnetic labeling is achieved with Anti-Immunoglobulin MicroBeads, Anti-Biotin MicroBeads, or Anti-Fluorochrome MicroBeads.
This indirect labeling strategy is useful for isolating untouched target cells. In this case, the unwanted cell types are incubated concurrently with a cocktail of primary antibodies conjugated to biotin, for example. Subsequently, cells are labeled with Anti-Biotin MicroBeads.
REAlease Technology is an indirect cell labeling method for positive selection of target cells. The REAlease Biotin Complex binds to the target cells. Labeling of the REAlease Biotin Complex with Anti-Biotin MicroBeads allows for magnetic isolation of these cells. Following cell separation, both MicroBeads and REAlease Biotin Complex can be gently removed, leaving the cells bead-and label-free. Enriched cells are then suitable for magnetic re-labeling and any application where label-free cells are essential.
Different technologies are available for the isolation of cells with nano-sized immunomagnetic beads: column-based MACS Technology by Miltenyi Biotec and column-free technologies by other manufacturers. A comparison is shown in the figure below.MACS Technology based on columns and nano-sized beads enables positive selection and depletion from any starting material including PBMCs, blood products, and dissociated tissues.
Magnetic separators in column-based and column-free systems generate a comparable magnetic force. What makes the difference, is the column. When the column is placed in a MACS Separator, the magnetic field gets amplified by 10,000-fold, due to the ferromagnetic spheres that are packed in the column.
Numerous data demonstrate that column-based cell isolation with MACS MicroBeads does not influence cell characteristics. In contrast, column-free technologies lead to alteration of cell characteristics, non-specific binding of immunomagnetic beads, and epitope saturation. Examples are shown in the figures below.
MACS MicroBeads are 50 nm in size and thus smaller than any other commercially available immunomagnetic beads for cell isolation. MACS MicroBeads are superparamagnetic, and therefore colloidal, i.e., they become magnetic when placed in a magnetic field. When removed from the magnetic field, they do not retain any residual magnetism. This feature of MACS MicroBeads is crucial, as particles with residual magnetism would aggregate quickly. In fact, aggregation can be observed with larger particles.MACS MicroBeads are biodegradable and non-toxic. Moreover, they have been designed to be compatible with any downstream application.
This option enables straightforward positive selection of target cells. Hundreds of reagents are available for the isolation of specific cell types and subsets. The strong magnetic field within a MACS Column allows for minimal cell labeling with nano-sized beads, thus preventing epitope saturation and non-specific labeling. Moreover, cells are not activated during the separation process.
MACS Cell Isolation Kits contain a cocktail of titrated antibodies and MACS MicroBeads for indirect magnetic labeling. They are the preferred choice if binding of antibodies to the target cells is not desired. Minimal labeling of unwanted cells with MACS MicroBeads avoids non-specific labeling of target cells, leaving the target cells truly untouched. In contrast, column-free methods based on nano-sized beads from other manufacturers require high concentrations of labeling reagents resulting in non-specific labeling of the target cell fraction (see figure below).
StraightFrom® MicroBeads allow magnetic isolation of various leukocyte subsets from different blood-derived starting materials by positive selection. In contrast to conventional methods, StraightFrom Technology does not require density gradient centrifugation, thus resulting in a simple and short protocol (see figure below).
MACSxpress® Technology enables fast isolation of cells directly from whole blood, omitting all centrifugation steps. This method is independent of columns. However, MACSxpress Beads are micro-sized and therefore allow for minimal labeling of unwanted cells, preventing non-specific labeling and activation of target cells. Non-target cells are removed by immunomagnetic depletion. Simultaneously, erythrocytes are sedimented to obtain target cell populations of exceptional purity.
REAlease® MicroBead Kits have been developed for positive selection of target cells from PBMCs. REAlease MicroBead Technology relies on recombinantly engineered antibody fragments instead of antibodies to label specific cell surface markers. The antibody fragments have a low affinity for cell surface epitopes. However, when the fragments are multimerized as a complex, they bind epitopes on target cells with high avidity and enable effective magnetic cell separation. REAlease Technology controls the multimer / monomer state of the fragments and thus triggers the release of monomerized antibody fragments from the cell surface after isolation. Ultimately, the isolated, positively selected target cells are free from antibody fragments and magnetic labels and are thus available for further separation steps (see figure below). Of course, the negative, i.e. non-labeled, cell fraction from this separation step is also available for further separation by positive selection (see figure below).
UltraPure MicroBeads have been particularly optimized for use with samples that contain large amounts of cell debris or low numbers of target cells. UltraPure MicroBeads greatly improve recovery and purity of the sorted population by specifically enriching viable target cells.