細胞分離 - MACS® テクノロジー

Questions & answers

What is MACS® Technology?

MACS® Technology is a fast and gentle method for the isolation of viable and functionally active cells by magnetic labeling.

MACS Technology is based on MACS MicroBeads, in combination with manual or automated MACS Separators and MACS Columns. When MACS Columns are placed in a MACS Separator, the MACS Column matrix provides a magnetic field strong enough to retain cells labeled with minimal amounts of magnetic material. Therefore, MACS MicroBeads used for labeling the cells can be so small and only a few are needed to separate a cell.

The magnetically labeled cells are separated over a MACS Column placed in a MACS Separator. They are retained on the column, while unlabeled cells pass through. These cells can be collected as the unlabeled fraction. The retained cells are eluted from the MACS Column after removal from the magnet. Excellent purity and recovery of both, labeled and unlabeled cells, can be obtained using MACS Technology

Which labeling strategies are possible?

Direct magnetic labeling
Direct labeling is a very fast one-step method. Highly specific monoclonal antibodies against cell surface molecules are directly conjugated to MACS® MicroBeads, resulting in low background and optimal separation results.

Indirect magnetic labeling
Indirect labeling is a two-step method. In a first step, cells are labeled with a primary antibody specific for a certain cell surface molecule. The primary antibody can be fluorochrome-conjugated, biotinylated, or unconjugated. In a second step, the magnetic labeling is performed with MACS® MicroBeads that target the primary antibody. This can be done with Anti-Fluorochrome MicroBeads, Anti-Biotin-MicroBeads, Streptavidin MicroBeads, or Anti-Isotype MicroBeads.

Which separation strategies are possible?

Positive selection
Positive selection means that the target cells are magnetically labeled with MACS® MicroBeads and directly separated as the positive cell fraction.
(see also "Columns for positive selection")

Depletion
Depletion means that the non-target cells are removed by magnetic labeling and separation.
A cocktail of antibodies for the depletion of several cell types is used to obtain a highly enriched subset of unlabeled cells.
Depletion can also be performed to remove one particular cell type from a cell population.
(see also " Columns for depletion")

Multi-parameter sorting
Multi-parameter sorting means that cells are isolated according to the expression of two markers. This can be done either by using MultiSort Kits or by combining depletion and positive selection.

MultiSort Kits
In general, MACS MicroBeads are not removed from the labeled cells (see also "Do MACS MicroBeads need to be removed prior to downstream applications?"). However, a number of MACS MultiSort MicroBeads has been developed for multiparameter purposes: After the first positive selection, MultiSort MicroBeads are released from the antibody. Thus, cells can subsequently be magnetically labeled for a second marker.

What are the most important parameters for magnetic labeling of cells with MACS® MicroBeads?

The three most important parameters are time, temperature and MicroBead concentration. These parameters are discussed below in further detail. Protocols for all MACS® Cell Separation Reagents are optimized for excellent results. Thus, it is strongly recommended to strictly follow the protocols in detail, given in the individual data sheets of the different MACS Cell Separation Products.

Time and temperature
The incubation time for magnetic labeling is 15 minutes for most MACS® Cell Separation Reagents. The incubation of the cells with MACS® MicroBeads should always be performed in the refrigerator, preferably at 4–8 °C. Higher temperatures and/or longer incubation times lead to non-specific cell labeling. Incubation on ice requires increased incubation time, otherwise cells might be insufficiently labeled.
However, for certain products incubation at room temperature or on ice may be required. In such cases, optimal incubation times are provided in the respective MACS® Cell Separation Reagent data sheet.

MicroBead concentration
Many MACS® MicroBeads are used at a 1:5 dilution: 107 cells are suspended in 80 µL of buffer and labeled with 20 µL of MicroBeads. Others are used at a 1:10 dilution: 107 cells are suspended in 90 µL of buffer and labeled by addition of 10 µL of MicroBeads. Always use dilutions as outlined in the protocol of the particular MACS® Cell Separation Reagent data sheet.

Do MACS® MicroBeads need to be removed prior to downstream applications?

Detachment of MACS® MicroBeads is not required.
MACS® MicroBeads are superparamagnetic particles of approx. 50 nm diameter. The non-toxic MicroBeads are composed of a biodegradable matrix. The scatter properties of magnetically labeled cells are not altered. Magnetic labeling with MACS® MicroBeads is compatible with downstream applications, such as flow cytometric or microscopic analysis, molecular biology applications, cell culture, or in vivo experiments.

Contact

Miltenyi Biotec K.K.
TEL +81 3 5646 8910
FAX+81 3 5646 8911
macs@miltenyibiotec.jp
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