MACS Handbook

Magnetic cell separation

1 Introduction

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.

2 Magnetic cell separation strategies using MACS® MicroBeads and Columns

At a glance: Separation strategies based on MACS MicroBeads and Columns
Separation strategyFeatures
Single separations
Positive selection of a target cell type
  • Target cell type is magnetically labeled and isolated from the cell suspension.
  • Unique marker for the target cell type is required.
Depletion of an unwanted cell type
  • Unwanted cell type is magnetically labeled and removed from the cell suspension.
  • Unique marker for the unwanted cell type is required.
Untouched isolation of a target cell type
  • All unwanted cells are magnetically labeled and removed from the cell suspension, leaving the target cells unlabeled, i.e., untouched.
  • No marker for target cells is required.
Sequential separations 
Depletion followed by positive selection
  • First step: Unwanted cells are magnetically labeled and removed from the cell suspension.
  • Second step: Target cells are magnetically labeled and isolated by positive selection. 
  • No unique marker for target cells is required if undesired cells expressing the positive-selection marker can be depleted effectively.
Two consecutive positive selections based on MultiSort MicroBeads and conventional MicroBeads
  • First step: Target cells are magnetically labeled with MultiSort MicroBeads and isolated. MultiSort MicroBeads are removed from the cells after separation.
  • Second step: Target cells are magnetically re-labeled with regular MACS MicroBeads and isolated.
  • Target cells are isolated using two different markers.
Two consecutive positive selections or positive selection followed by depletion based on REAlease® Technology
  • First step: Target cells are labeled with REAlease Biotin Complex and Anti-Biotin MicroBeads and separated. Entire labeling complex can be removed from the cells after separation.
  • Second step: Target cells or residual unwanted cells within the enriched target cell fraction can be re-labeled with REAlease Technology or conventional MACS MicroBeads for positive selection or depletion, respectively.
  • Epitope of the marker used for positive selection becomes completely available for a second round of positive selection.
  • No unique marker for target cells is required.
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2.1 Positive selection of a target cell type

Positive selection of a target cell type
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Positive selection of a target cell type. In the example shown, target cells are indirectly magnetically labeled.

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. Positive selection can be performed by direct or indirect magnetic labeling. Specific MACS MicroBeads are available for the positive selection of numerous cell types and cell subsets.

For details about different options to label cells, see chapter Labeling strategies below.

2.2 Depletion of an unwanted cell type

Depletion of an unwanted cell type
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Depletion of an unwanted cell type. In the example shown, undesired cells are directly magnetically labeled.

To remove a certain cell type from a mixture of cells, the unwanted cells are magnetically labeled. During separation, the unlabeled target cells are collected in the flow-through fraction whereas the unwanted cell type is retained within the column. Optionally, the retained cells can be eluted after removal of the column from the separator (not shown).

2.3 Isolation of an untouched target cell type

Isolation of an untouched target cell type
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Isolation of an unlabeled, i.e., untouched target cell type. In the example shown, unwanted cells are indirectly magnetically labeled.

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.

2.4 Sequential separations

If a certain desired cell type or subset does not express a single unique marker, it is not possible to isolate these cells in a single positive selection step. Instead a combination of two consecutive separations based on different markers can be applied.
2.4.1 Depletion followed by positive selection
Depletion followed by positive selection
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Depletion of unwanted cells, followed by positive selection of a target cell type.

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.

2.4.2 Two consecutive positive selections
Cell separation with MACS MultiSort MicroBeads
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Cell separation with MACS MultiSort MicroBeads. Following the first positive selection, the MultiSort MicroBeads are removed from the cells, thus allowing for a second positive selection.

MACS MultiSort MicroBeads enable cell isolation by two sequential positive selection steps based on two different markers. For the first positive selection, cells are labeled with MACS MultiSort MicroBeads specific for the first marker. After elution from the column, the cells are incubated with MultiSort Release Reagent, which enzymatically removes the MultiSort MicroBeads from the cells. For the second positive selection, the target cells are magnetically labeled with MACS MicroBeads directed against the second marker and isolated on the column.
2.4.3 Two consecutive positive selections or positive selection followed by depletion
Principle of REAlease Technology
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Principle of REAlease Technology. 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 for further cell separation steps.

REAlease Technology allows for magnetic cell isolation by positive selection of target cells and subsequent removal of any beads and labels from the cells. The technology relies on recombinantly engineered antibody fragments instead of whole antibodies for cell surface labeling. Monomeric fragments have a low affinity for cell surface markers. However, when multimerized as part of the REAlease Complex, the fragments bind markers with high avidity. Anti-Biotin MicroBeads bound to the REAlease Complex facilitate isolation of the target cells based on MACS Columns and Separators (see figure). When the column is placed in the magnetic field of the separator, unlabeled cells flow through, whereas the target cells are retained on the column. Subsequently, labeled target cells are eluted from the column using REAlease Bead Release Reagent, which removes Anti-Biotin MicroBeads from the cells. Following elution, there are two options to isolate subsets from this population: I) Disruption of the REAlease Complex by adding REAlease Release Reagent leads to monomerization and spontaneous dissociation of the antibody fragments from the cell surface. Cells are then label free and suitable for magnetic re-labeling with REAlease Technology. II) Alternatively, eluted cells can be directly labeled with MACS MicroBeads.

3 Magnetic labeling strategies using MACS MicroBeads

3.1 Direct magnetic labeling

Direct magnetic labeling
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Direct magnetic labeling of cells with MicroBeads.

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.

3.2 Indirect magnetic labeling

Combination of primary antibodies and MicroBeads
Indirect magnetic labeling
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Indirect magnetic labeling with primary antibodies and MicroBeads.

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

REAlease Technology
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Indirect magnetic labeling with REAlease Technology.

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.

4 Comparison of different magnetic cell separation methods based on nano-sized beads

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.

Comparison between column-based MACS MicroBead Technology and column-free magnetic cell separation technologies. Human PBMCs were either labeled with MACS CD3 MicroBeads for the isolation of T cells with a MACS Column or with other nano-sized beads for column-free isolation of the same cell type. Scanning electron microscopy showed (A) no visible labeling on the cell surface after isolation with MACS MicroBeads and MACS Columns, whereas (B) excessive labeling became obvious (indicated by arrows) after isolation with column-free technology from another manufacturer.

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5 MACS Columns

5.1 MACS Columns enable gentle cell isolation

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. 

MACS Columns
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Detailed view of a MACS Column. Small ferromagnetic spheres contained in the column amplify the magnetic field generated by a MACS Separator. The spheres have a cell-friendly coating.

Due to the strong amplification of the magnetic field, minimal labeling of cells with nano-sized superparamagnetic beads suffices to retain target cells in the column. Labeled cells hover between the ferromagnetic spheres while unlabeled cells pass freely through the column. The columns can be washed thoroughly to remove cell debris and undesired cells. MACS Columns thus provide gentle conditions for cell separation. Besides this, MACS Cell Separation is easy to accomplish and compatible with any downstream application, including cell sorting, flow cytometry analysis, cell culture, functional assays, and molecular analysis.
Schematic view of a MACS Column
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Schematic view of a MACS Column. Cells move freely between the spheres inside the column and are only retained by magnetic forces.

Amplification of the magnetic force within the MACS Column
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Amplification of the magnetic force within the MACS Column allows cell separation with minimal labeling. Without the use of a MACS Column, extensive labeling or large beads are needed for an adequate magnetic retention (A). Only the amplification of the magnetic force by MACS Columns ensures effective cell retention with minimal labeling using small beads (B).

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.

Column-based MACS Technology requires only minimal cell labeling
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Column-based MACS Technology requires only minimal cell labeling and thus preserves cellular integrity and characteristics. Human PBMCs were labeled with MACS MicroBeads for the isolation of monocytes with a MACS Column (A) or with other nano-sized beads for column-free isolation of the same cell type (B). Scanning electron microscopy showed no visible labeling on cells or alteration of the cells’ appearance when the cells were isolated with MACS MicroBeads. In contrast, cells isolated column-free with other nano-sized beads showed massive labeling (indicated by arrows) and an altered visual appearance.

Minimal cell labeling with MACS MicroBeads avoids epitope blocking. 
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Minimal cell labeling with MACS MicroBeads avoids epitope blocking. CD138+ cells were isolated from whole blood with CD138 Whole Blood MicroBeads and a MACS Column (upper right) or other nano-sized beads and a column-free method (lower right). The original whole blood sample (left) and isolated cells (right) were stained for CD45, CD19, and CD138 and analyzed by flow cytometry. Only the cells isolated with MACS MicroBeads could be analyzed reliably for CD138, indicating that a sufficient number of epitopes remained available for fluorescent staining on magnetically labeled cells.

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5.2 MACS Columns at a glance

For details about the different MACS Columns, see the page MACS Columns at a glance on our website.

6 MACS MicroBeads

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, non-toxic, and tested according to the International Standard (ISO 10993) and USP guidelines for biocompatibility. Moreover, they have been designed to be compatible with any downstream application.
MACS MicroBeads are conjugated to highly specific antibodies against a certain cell surface antigen. They bind specifically to cells expressing this antigen, thus preventing non-specific labeling. Due to the low labeling concentrations and their small size, MACS MicroBeads do not lead to activation of target cells as demonstrated in the figure below.
MACS MicroBeads do not activate target cells, for example, B cells.
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MACS MicroBeads do not activate target cells, for example, B cells. Human B cells were enriched using MACS CD19 MicroBeads or a column-free positive selection method from another manufacturer. Subsequently, cells were cultured for 7 days in the presence or absence of the B cell stimulation reagents CD40-Ligand/Anti-His antibody and IL-4. Activation markers (CD69, CD80, and CD86) were measured by flow cytometry directly after cell isolation and after cultivation with and without stimulation. MACS MicroBeads did not alter the status of the target cells, whereas the column-free method led to the activation of B cells in the absence of stimulation reagents.

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6.1 Cell isolation from single-cell suspensions such as dissociated tissue

6.1.1 MACS MicroBeads and MicroBead Kits: positive selection of target cells based on specific markers

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. 

  • The gentlest, least manipulative positive selection method available
  • Preservation of cell functionality due to optimal labeling
  • Labeled cells are ready for downstream applications as beads are biodegradable
 For details about positive selection with MACS MicroBeads, see chapter Positive selection of a target cell type above.
6.1.2 MACS Cell Isolation Kits: isolation of pure, truly untouched cells by depletion of non-target cells

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).

  • High purity and recovery rates
  • Fully compatible with any downstream application
  • No non-specific labeling of target cells
MACS Cell Isolation Kits enable isolation of truly untouched cells
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MACS Cell Isolation Kits enable isolation of truly untouched cells. Monocytes were enriched by depletion of unwanted cells using the MACS Monocyte Isolation Kit II, human or a column-free kit for human monocyte isolation from another manufacturer. Staining of monocytes (red) and nano-sized beads (green) after isolation showed non-specific labeling of the target cells when using column-free kits, while MACS Technology provided trulyuntouched cells.

For details about depletion of non-target cells, see chapter Isolation of an untouched target cell type above.

6.2 Cell isolation directly from blood products

6.2.1 StraightFrom® Technology: cell isolation directly from blood products without density gradient centrifugation

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). 

  • Cell isolation directly from whole blood, buffy coat, and leukocyte reduction system chamber (LRSC)
  • Isolated target cells are immediately ready for any downstream application
  • Simple protocol with only few handling steps
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Comparison of the StraightFrom MicroBeads protocol  with conventional protocols.

6.2.2 MACSxpress® Technology: Isolation of untouched target cells directly from whole blood

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.

  • Isolated cells from whole blood within 20 minutes
  • No density gradient centrifugation
  • High reproducibility due to minimal sample handling
Basic principle of MACSxpress Technology.
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Basic principle of MACSxpress Technology.

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6.3 Isolation of label-free cells and processing of challenging samples

6.3.1 REAlease® Technology: Isolation of bead- and label-free cells

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).

  • Bead-free cells: suited for second round of magnetic labeling
  • Label-free cells: the epitope of a marker becomes completely available again
  • Recombinantly produced: lot-to-lot consistency allows for reproducible results
 For details about REAlease Technology, see chapter Two consecutive positive selections or positive selection followed by depletion above.
REAlease Technology
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Isolation of CD56+CD3 NK cells using REAlease Technology. REAlease CD56 MicroBeads were used for the enrichment of CD56+ cells from PBMCs. After removal of REAlease MicroBeads, the CD3+ cell fraction was depleted from the CD56+ cell population using MACS CD3 MicroBeads. This resulted in a population of CD56+CD3 NK cells with a purity of 98%. No difference between unseparated and enriched cells was observed with regard to the levels of the activation markers CD69 and CD25 .

REAlease Technology
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Isolation of label-free CD4+ and CD8+ cells from a single sample based on REAlease Technology. CD8+ cells were isolated from PBMCs using REAlease CD8 MicroBeads. The negative fraction from this separation step was used as starting material for the isolation of CD4+ cells with REAlease CD4 MicroBeads. Both cell populations were isolated with yields higher than 80% and purities exceeding 98%.

6.3.2 UltraPure MicroBeads: Isolation of cells from debris-rich samples

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.

  • Optimized formulation to minimize debris
  • High cell purity, even from challenging starting materials
  • As easy to use as the conventional MACS MicroBeads
UltraPure MicroBeads
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Isolation of CD34+ cells from a debris-rich sample. CD34+ cells were isolated with the column-based CD34 MicroBead Kit UltraPure (upper plots) or with a column-free positive selection method from another manufacturer (lower plots). The cell population purified with MACS MicroBeads UltraPure showed significantly reduced amounts of debris compared to the column-free method.

7 MACS Separators

MACS Cell Separation can be performed manually or in an automated fashion.

For details about manual separators, see the page MACS Manual Separators for magnetic cell isolation on our website
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8 Automation

MACS Technology offers a broad range of options for automated cell isolation, depending on the desired sample volume and throughput. Automation makes for a high level of standardization and thus ensures reliable, user-independent results.

8.1 MultiMACS™ Cell24 Separator Plus – processing of large sample numbers or volumes

For high-throughput settings, in which large sample numbers or volumes need to be processed, the semi-automated MultiMACS™ Cell24 Separator Plus is the right choice. This instrument can be used in combination with MACS MicroBeads and MACS Cell Isolation Kits to isolate a wide variety of cell types or to deplete specific cell types. The MultiMACS Cell24 Separator Plus enables cell separation from up to 24 samples in parallel.
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MultiMACS™ Cell Separator Family: Automation meets parallel cell separation

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8.2 autoMACS® Pro Separator – full automation of cell labeling and isolation

Completely automated cell labeling and isolation are made possible with the autoMACS® Pro Separator. Up to six samples can be processed in one go. The instrument is compatible with hundreds of MACS Cell Separation Reagents for an easy and fast isolation of virtually any cell type from any species. 
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autoMACS Pro Separator: The gold standard in automated cell separation 

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8.3 MultiMACS X – full automation and high-throughput sample processing

The MultiMACS X combines true walk-away convenience with high-throughput sample processing. It combines all the benefits of the MultiMACS Cell24 Separator Plus with a customized liquid handler for precise pipetting and sample transfer. Parallel processing ensures a high level of reproducibility.
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MultiMACS Cell Separator Family: Automation meets parallel cell separation 


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