Gold standard in cell separation

Transcription

1 MACS Technology Gold standard in cell separation Consistent, high-quality separations Easy handling Compatible with downstream applications Innovative products From lab bench to clinical research applications

2 From lab bench to clinical research applications From small samples to samples ranging up to cells by using columns with different cell loading capacity and their suitable separators Automated cell separation: The automacs Pro Separator allows standardization of frequent cell separations. Sterile separations in a closed system: The CliniMACS Plus Instrument in combination with GMP reagents opens new opportunities for clinical research. Compatible with downstream applications Gentle on cells: small MicroBeads in combination with MACS Column Technology allow the purification of viable and functionally active cells. No influence on analysis: small MicroBeads do not influence flow cytometric or microscopic analysis. Suitable for cell culture and in vivo experiments: MicroBeads are biodegradable, the short procedure and gentle column technology avoid mechanical stress. Molecular biology experiments: limited amount of small MicroBeads avoid any influence on further experiments. Spleen cells before separation Isolated CD4 + CD25 + T cells CD25-PE CD25-PE automacs Pro Separator Walk-away cell sorting of multiple samples CD4 + CD25 + regulatory T cells isolated from mouse spleen using MACS Technology CD133 + cells isolated with MACS Technology became adherent and CD133 during cultivation but gave rise to non-adherent CD133 + cells budding from the adherent cell surface by asymmetric cell division Innovative products Continuous product development with focus on current research and clinical research needs CliniMACS Plus Instrument The CliniMACS System a closed and sterile system for separation of large cell samples T cells: isolation of regulatory T cells, naive and memory/effector cells, Th2 cells, or even antigen-specific T cells according to their secretion of cytokines Stem cells: CD133 the marker for isolation of hematopoietic and nonhematopoietic stem cells Dendritic cells: unique kits for isolation of myeloid and plasmacytoid human dendritic cells and mouse dendritic cell subsets Plasmacytoid blood dendritic cells isolated with MACS Technology and stained with May-Grünwald- Giemsa

3 MACS Technology Gold standard in cell separation The advantages Consistent, high quality separations Optimal purity and recovery due to highly specific reagents combined with efficient MACS Column Technology Frequent cells such as T, B, NK cells and monocytes, but also rare cells like stem cells, antigen-specific T cells, and dendritic cell subsets For any cell type: over 200 reagents for the direct isolation of human, mouse, rat, and non-human primate cells; any other cell type can be isolated using indirect labeling with MicroBeads Reliable and consistent results: based on extensive experience in cell separation systems and rigorous quality control Easy handling Convenient procedure: easy-to-use system, applicable in every lab Fast results: cells can be purified in up to 30 minutes for positive selection or depletion due to short incubation time and efficient MACS Column Technology; no time-consuming bead detachment necessary For positive selection and depletion strategies: both labeled and unlabeled fractions can be obtained with excellent purity and recovery Prepackaged starting kits available: consisting of a magnet and stand, columns and reagents Mouse spleen CD4 + T cells isolated using an LS Column and a MidiMACS Separator CD4 + T cells isolated using the automacs Separator CD3ε-APC Human bone marrow Bone marrow cells before separation Isolated CD34 + cells MiniMACS Separator Basic tool for the lab: MiniMACS Separator with an MS Column CD34-FITC CD34-FITC CD8-PE CD38-PE CD38-PE QuadroMACS Separator For the simultaneous separation of 4 samples

4 MACS Technology Gold standard in cell separation MACS Technology MACS Technology has become the standard method for cell separation. Numerous publications have proven its versatility for multiple applications: cell separations with consistent high-quality results from lab bench to clinical applications, from small to large scale, from frequently occurring cells to rare cells and sophisticated subsets. Miltenyi Biotec provides researchers worldwide with the tools for high-quality separations. MACS Cell Separation is based on the use of MACS MicroBeads, MACS Columns, and MACS Separators. Cells can easily be purified or depleted in less than 30 minutes. Magnetic labeling Gentle to cells; minimal influence on downstream experiments The principle MACS Technology is based on MACS MicroBeads, MACS Separators, and MACS Columns. MACS MicroBeads are superparamagnetic particles of approximately 50 nanometers in diameter. They are composed of a biodegradable matrix, and it is therefore not necessary to remove them from cells after the separation process. Usually, MACS MicroBeads do not alter structure, function, or activity status of labeled cells and are not known to interfere with subsequent experiments. Magnetic separation MACS Column Technology provides a high-gradient magnetic field. Gentle to cells Thorough rinsing procedure High recovery Untouched isolation by depletion of specific cells N S MACS Technology takes place within MACS Columns. When a MACS Column is placed in a MACS Separator, a strong permanent magnet, a high-gradient magnetic field is induced on the column matrix strong enough to retain cells labeled with minimal amounts of MACS MicroBeads. Unlabeled cells pass through and can be collected; labeled cells are released after removal of the column from the magnet. Thus, with MACS Technology both labeled and unlabeled cell fractions can easily be isolated with high purity. The entire procedure of positive selection or depletion takes less than 30 minutes, and cells can immediately be used for further experiments. Elution of the labeled cell fraction Optimal results even for rare cells by using positive selection CD133/2-PE Anti-Biotin-PE CD34-FITC Electron micrographs on this page with courtesy of Prof. Peter Groscurth, Institute of Anatomy, University of Zürich, Switzerland.

5 MACS Magnetic Labeling Direct magnetic cell labeling Indirect magnetic cell labeling Direct labeling is the fastest way of magnetic labeling. It requires only one labeling step since the specific antibody is directly coupled to the magnetic particle. Direct labeling reduces the number of washing steps and, therefore, avoids unnecessary cell loss. Miltenyi Biotec provides highly specific monoclonal antibodies for low background and optimal separation, targeted against surface markers of cells from various species such as human, mouse, rat, and non-human primates. Fluorochrome-conjugated MACS Antibodies can be added for the purpose of analyzing magnetically separated cell fractions by flow cytometry or fluorescence microscopy. Indirect labeling is performed if no direct MicroBeads for the cell type of interest are available. Almost any monoclonal or polyclonal antibody targeting any cell type from any species can be used for indirect labeling. Cells are labeled with a primary antibody that is unconjugated, biotinylated, or fluorochrome-conjugated. In a second step, magnetic labeling is performed by using Anti-Immunoglobulin, Anti-Biotin, Streptavidin, or Anti-Fluorochrome MicroBeads, respectively. A cocktail of antibodies can also be used to isolate or deplete a number of cell types concurrently. Since indirect labeling amplifies the magnetic label, it may be the method of choice for magnetic separation according to dimly expressed antigens. Anti-Immunoglobulin MicroBead Anti-Biotin MicroBead Antibody MicroBead Streptavidin MicroBead Streptavidin MicroBead Biotinylated antibody Fluorochrome-conjugated antibody Anti-Fluorochrome MicroBead

6 MACS Separation Strategies Positive selection strategy Untouched isolation Positive selection means that the desired target cells are magnetically labeled and isolated as the magnetically retained cell fraction. A positive selection strategy should be considered for: excellent purity, especially for rare cell enrichment, excellent recovery, and fast results. Positive selection is the most direct and specific way to isolate the target cells from a heterogenous cell suspension. Binding of antibodies (which are part of the MicroBeads) to the cell surface does not affect viability or function of the cells. Both fractions, labeled and unlabeled, can be recovered and used. Due to their composition of iron oxide and polysaccharide, MicroBeads are biodegradable and typically disappear after a few days in culture. Untouched isolation is performed by depletion of undesired cells. Non-target cells are magnetically labeled and eliminated from the cell mixture. The non-magnetic, untouched cell fraction contains the target cells. Untouched isolation should be considered: for removal of unwanted cells, if no specific antibody is available for target cells, if binding of the antibody to the target cells is not desired, or for subsequent isolation of a cell subset by means of positive selection. For many different cell types Miltenyi Biotec offers optimized MACS Cell Isolation Kits containing pre-titrated cocktails of antibodies directed against non-target cells. Magnetic labeling Cells of interest are magnetically labeled with MACS MicroBeads. Magnetic labeling Non-target cells are magnetically labeled with a biotinylated antibody cocktail and Anti-Biotin MicroBeads. Magnetic separation Cells are separated in a MACS Column placed in a MACS Separator. The flow-through fraction can be collected as negative fraction depleted of the labeled cells. Magnetic separation Undesired cells are retained in a MACS Column placed in a MACS Separator. The target cells pass through the column and are collected as the enriched, unlabeled cell fraction, depleted of non-target cells. Elution of the labeled cell fraction The column is removed from the separator. The retained cells are eluted as the enriched, positively selected cell fraction.

7 MACS Separation Strategies Sequential sorting (1): Depletion followed by positive selection Cell subsets can be isolated by first depleting the non-target cells and then positively selecting the cell subsets of interest. This strategy is useful if undesired cells in the cell suspension express the same antigen which is used for positive selection of the target cells. For isolation of extremely rare cells, it also can be useful first to deplete non-target cells from the suspension. Positive selection can then be carried out with the pre-enriched fraction to obtain very pure cells. 1st magnetic labeling Non-target cells are magnetically labeled with a biotinylated antibody cocktail and Anti-Biotin MicroBeads. Sequential sorting (2): MultiSort strategy With MACS MultiSort Kits high numbers of cells, characterized by multiple cell surface markers, can be sorted easily. Even rare cells can be enriched very efficiently. Multi-parameter sorting with MACS MultiSort Kits allows sequential positive selections of cells. The target cells are first labeled with MACS MultiSort MicroBeads and positively selected for the first parameter. Then the cells are incubated with the MultiSort Release Reagent which enzymatically removes the MicroBeads from the antibodies. In the next step, the target cells are magnetically labeled with MACS MicroBeads directed against a subset marker to be positively selected a second time. 1st magnetic separation Undesired cells are retained in a MACS Column placed in a MACS Separator while the unlabeled cells pass through. 1st magnetic labeling Cells of interest are magnetically labeled with MultiSort MicroBeads. 1st magnetic separation Target cells are magnetically isolated by positive selection. 2nd magnetic labeling Target cells are magnetically labeled with MicroBeads according to a subset marker. Release of magnetic particles MultiSort MicroBeads are enzymatically released. 2nd magnetic separation Target cells are retained in the column while unlabeled cells pass through. After the column is removed from the separator, the target cells are eluted as the enriched, positively selected cell fraction. 2nd magnetic labeling Cell subset of interest is labeled with MACS MicroBeads according to a second marker. 2nd magnetic separation Target cells are positively selected a second time.

8 Miltenyi Biotec offers a wide variety of products for magnetic cell isolation in basic research as well as for clinical-grade cell separations providing the perfect tools for translational research. The continuously growing portfolio also includes instrumentation and numerous antibodies for flow cytometry, as well as products for sample preparation, cell activation and expansion, and molecular analysis. To find out more, visit our homepage at Germany/Austria/ Switzerland Miltenyi Biotec GmbH Friedrich-Ebert-Straße Bergisch Gladbach Germany Phone Fax [email protected] USA/Canada Miltenyi Biotec Inc Lindbergh Street Auburn, CA 95602, USA Phone 800 FOR MACS Phone Fax [email protected] Australia Miltenyi Biotec Australia Pty. Ltd. Unit 16A, 2 Eden Park Drive North Ryde, NSW 2113 Australia Phone Fax [email protected] Benelux Miltenyi Biotec B.V. Postbus 85183, 3508 AD Utrecht The Netherlands [email protected] Customer service Netherlands Phone Fax Customer service Belgium Phone Fax Customer service Luxembourg Phone Fax China Miltenyi Biotec Trading (Shanghai) Co., Ltd. Shanghai Office Fareast International Plaza A Rm. 2301, No. 319 Xianxia Rd. Shanghai , P.R. China Phone Fax [email protected] France Miltenyi Biotec SAS 10 rue Mercoeur Paris, France Phone Fax [email protected] Italy Miltenyi Biotec S.r.l. Via Persicetana, 2/D Calderara di Reno (BO) Italy Phone Fax [email protected] Japan Miltenyi Biotec K.K. Nittsu-Eitai Building 5F Fuyuki, Koto-ku, Tokyo , Japan Phone Fax [email protected] Singapore Miltenyi Biotec Asia Pacific Pte Ltd. 100 Beach Road #28-06 to Shaw Tower Singapore Phone Fax [email protected] Spain Miltenyi Biotec S.L. C/Luis Buñuel 2 Ciudad de la Imagen Pozuelo de Alarcón (Madrid), Spain Phone Fax [email protected] United Kingdom Miltenyi Biotec Ltd. Almac House, Church Lane Bisley Surrey GU24 9DR, UK Phone Fax [email protected] Miltenyi Biotec provides products and services worldwide. Visit to find your nearest Miltenyi Biotec contact. The CliniMACS System components: Instruments, Reagents, Tubing Sets, and PBS/EDTA Buffer are manufactured and controlled under an ISO certified quality system. In Europe, the CliniMACS System components areavailable as CE-marked medical devices. In the USA, the CliniMACS System components including the CliniMACS Reagents are available for use only under an approved Investigational New Drug (IND) application or Investigational Device Exemption (IDE). CliniMACS MicroBeads are for research use only and not for use in humans. automacs and CliniMACS are registered trademarks of Miltenyi Biotec GmbH. MiniMACS and QuadroMACS are trademarks of Miltenyi Biotec GmbH. Unless otherwise specifically indicated, Miltenyi Biotec products and services are for research use only and not for therapeutic or diagnostic use. Copyright 2010 Miltenyi Biotec GmbH. All rights reserved.