A high performance system in which cells are immunomagnetically labeled & bound to a magnetic column all species all cell types positive or negative

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1 A high performance system in which cells are immunomagnetically labeled & bound to a magnetic column all species all cell types positive or negative selection

2 TM Immunomagnetic or Positive Cell ALL SPECIES ALL CELL TYPES and More! Human, rat or non-human primate cells are crosslinked to magnetic dextran iron particles using TAC (Tetrameric Antibody Complexes; Figure 1). TAC are comprised of two mouse IgG 1 monoclonal antibodies (shown in red & blue in Figure 1), held in tetrameric array by two rat anti-mouse IgG 1 monoclonal antibody molecules (shown in yellow in Figure 1). One mouse antibody molecule recognizes the cell surface antigen and the other recognizes the dextran on the magnetic colloid/nanoparticle. The cell suspension is then passed through a high-gradient column of stainless steel mesh which is placed in a magnet. and More! negative selection The magnetically labeled unwanted cells bind to the column while the unlabeled enriched cells pass through untouched leaving them ready for further study. Figure 2. StemSep method for of cells Add cocktail of tetrameric antibody complexes, incubate, then add magnetic colloid and incubate Unwanted cells are labeled Desired cells are unlabeled human, rat, or non-human primate Figure 1. Schematic drawing of StemSep TAC magnetic labeling of human, rat or non-human primate cells Cell Cell positive selection The magnetically labeled cells of interest bind to the column while the unlabeled cells pass through. Cells of interest are collected from the positive selection column according to manufacturer s instructions. The extremely tiny magnetic nanoparticles are completely FACS-compatible, and thus do not interfere with further studies Figure 3. StemSep method for Positive of cells Add StemSep positive selection cocktail, incubate, then add magnetic colloid and incubate Cells of interest Unwanted cell Dextran Tetrameric Antibody Complex Magnetic Colloid mouse Mouse cells are magnetically labeled indirectly using a cocktail of biotinylated antibodies directed against cell surface markers on the unwanted cells (Figure 4). Cells are then crosslinked to magnetic dextran iron particles using TAC (Tetrameric Antibody Complexes). TAC are comprised of two mouse IgG 1 monoclonal antibodies held in tetrameric array by two rat anti-mouse IgG 1 monoclonal antibody molecules. One mouse antibody molecule recognizes biotin and the other recognizes dextran on the magnetic nanoparticle. The cell suspension is then passed through a high-gradient column of stainless steel mesh (Figure 5). The magnetically labeled unwanted cells bind to the column while the unlabeled enriched cells pass through. Figure 4. Schematic drawing of StemSep TAC magnetic labeling of mouse cells Cell Biotinylated Antibody Biotin Dextran Tetrameric Antibody Complex negative selection Figure 5. StemSep method for of mouse cells Add cocktail of biotinylated antibodies Magnetic Colloid Pass cell suspension through column placed in magnet Cells are ready for separation. Follow directions recommended by positive selection column manufacturer Unwanted cells are labeled Desired cells are unlabeled Wash Unwanted cells (labeled) bind to column Magnet Magnet Cells of interest bind to column Add tetrameric antibody complexes, incubate, then add magnetic colloid and incubate Collect enriched cells Recovered cells have never been labeled with antibody and are ready for future studies Unwanted cells pass through Pass cell suspension through column placed in magnet Unwanted cells (labeled) bind to column Collect enriched cells PLUS for any species: try a custom cocktail or use the stemsep pe or biotin tac with your own antibodies! Recovered cells have never been labeled with antibody and are ready for future studies

3 TM Equipment & Reagents Magnets Red Magnet with Stand This device is designed to perform one gravity or pump feed separation with a StemSep 0.1 or 0.3 negative selection column. Depending on the column size, a range of x 10 7 cells can be loaded per column. Blue Magnet with Stand and Pump This device is designed to perform up to four simultaneous separations with the following StemSep negative selection column sizes: 0.3, 0.5 and 0.6. Depending on the column size, a range of 2 x x 10 9 cells can be loaded per column. This magnet and stand can accommodate only pump feed columns. Green Magnet with Stand This device is designed to perform one gravity or pump feed separation with the following StemSep negative selection column sizes: 0.1, 0.3, 0.5 and 0.6. Depending on the column size, a range of x 10 9 cells can be loaded per column. Columns Black Magnet with Stand and Pump This device is designed to perform a single pump feed separation with a 1.0 bulk column. Optimally, cells can be processed with this column. This magnet and stand can also accommodate the following pump feed columns: 0.3, 0.5, 0.6 (and 1.0 ) giving a range of 2 x x cells per separation G P G P G P G P Columns are available in five sizes (optimal cell number/column*) 0.1 diameter (10 6 cells), 0.3 diameter (5 x 10 7 cells), 0.5 diameter (10 8 cells), 0.6 diameter (5 x 10 8 cells) and 1.0 diameter (10 10 cells) High-gradient magnetic columns comprised of stainless steel mesh High capacity columns designed for negative selection Pump (P) or gravity (G) feed Cell range from 10 5 to 1.5 x cells depending on the column Positive selection reagents are designed for use with commercially available magnetic positive cell selection columns * Optimum cell number/column can be doubled when using ficolled cells Reagents - Ultimate in Flexibility & Customization Positive or Human, mouse, rat, non-human primate, & more Compatible with other immunomagnetic column-based systems (0.5 Tesla magnet) including manual and automated systems Incorporate any mouse IgG 1 monoclonal antibody to create a customized TAC** cell separation reagent Use any biotinylated or PE-conjugated antibody with anti-biotin or anti-pe TAC** FACS-compatible magnetic nanoparticles A wide selection of antibodies are available for custom separations. Please contact us to discuss your needs See next pages for composition of human (negative & positive selection) & mouse (negative selection) standard cocktails as well as suggested combinations of antibodies for particular cell subsets. ** Tetrameric Antibody Complexes

4 StemSep Cocktails - target unwanted cells Hematopoietic Stem/Progenitor Cell Hematopoietic Progenitor Cocktail CD5, CD11b, CD45R (B220),, Neutrophils (7-4) Double Thymocyte T Cell Cocktail plus antibodies against: NK Cells, CD4, CD8, CD25 T Cell CD11b, CD45R (B220) γδ T Cell T Cell Cocktail plus antibodies against NK cells, TCRαβ NK Cell B Cell CD4, CD8, CD11b, Ly-6G (Gr-1), TER119 CD5, CD22, F4/80, CD4 T Cell CD8 T Cell CD8, CD11b, CD45R (B220) CD4, CD11b, CD45R (B220), NK cells (DX5), NaÏve CD4 T Cell NaÏve CD8 T Cell CD4 T Cell Cocktail plus antibodies against: NK Cells, CD44 CD8 T Cell Cocktail plus antibodies against: CD44 Memory CD4 T Cell Memory CD8 T Cell CD4 T Cell Cocktail plus antibodies against: NK Cells, CD62L CD8 T Cell Cocktail plus antibodies against: CD62L Resting CD4 T Cell Resting CD8 T Cell CD4 T Cell Cocktail plus antibodies against: CD25 CD8 T Cell Cocktail plus antibodies against: CD25 * Biotinylated antibodies in standard negative selection cocktails are shown in blue. Biotinylated antibodies suggested for addition to a standard cocktail to enrich a particular cell subset are shown in red (custom cocktail). Dendritic Cell CD2, CD3, CD19,, Neutrophils (7-4)

5 TM Immunomagnetic Human Cell Composition of Cocktails* StemSep Cocktails - target unwanted cells StemSep Positive Cocktails - target desired cells Hematopoietic Stem/Progenitor Cell Primitive Hematopoietic Progenitor Cocktail Hematopoietic Progenitor Cocktail CD2, CD3, CD14, CD16, CD19, CD24, CD36, CD38, CD45RA, CD56, CD66b, CD2, CD3, CD14, CD16, CD19, CD24, CD56, CD66b, CD34 Lymphoid Progenitor Myeloid Progenitor T Cell B Cell NK Cell Dendritic Cell Monocyte Granulocyte CD14, CD16, CD19, CD56, CD3 CD2, CD3, CD14, CD16, CD56, CD19 CD3, CD4, CD14, CD19, CD66b CD56 CD3, CD14, CD16, CD19, CD34, CD56, CD66b, CD2, CD3, CD19, CD56, CD66b, CD14 CD2, CD3, CD14, CD19, CD56, Eosinophil Neutrophil Basophil CD2, CD14, CD16, CD19, CD56, Use polymorphonuclear (PMN) cell-rich fraction Suggested custom cocktail: CD23, IgE Use polymorphonuclear (PMN) cell-rich fraction CD2, CD3, CD14, CD15, CD16, CD19, CD24, CD34, CD36, CD45RA, CD56, γδ T Cell αβ T Cell T cell cocktail plus: TCRαβ T cell cocktail plus: TCRγδ Other Enrichment Cocktails CD4 T Cell CD8 T Cell CD8, CD14, CD16, CD19, CD56, CD4 CD4, CD14, CD16, CD19, CD56, CD8 CD4 CD25 Refer to RosetteSep & EasySep protocol antigen stimulation NaÏve CD4 T Cell NaÏve CD8 T Cell CD8, CD14, CD16, CD19, CD45RO, CD56, CD8T cell cocktail plus: CD45RO antigen stimulation Effector CD4 T Cells Epithelial Tumor Cell CD2, CD14, CD16, CD38, CD45, CD66b, Human Cell Enrichment from Mouse/Human Chimeras (anti-mouse) CD45, TER119 CML De-Bulking CD2, CD3 CD14, CD15, CD16, CD19, CD24, CD56, CD66b, IgE, Effector CD8 T Cells Th1 Th2 CD4T cell cocktail plus one or more of: CD124 (IL-4R), CCR4, T1/ST2 CD4T cell cocktail plus: IL-18rα Depletion Cocktails CD3 T Cell Depletion CD3 CD8 T Cell Depletion Memory CD4 T Cell Resting CD4 T Cell Memory CD8 T Cell Resting CD8 T Cell CD4T cell cocktail plus: CD45RA CD4T cell cocktail plus one or more of: CD25, CD69, HLA-DR CD8T cell cocktail plus: CD45RA CD8T cell cocktail plus one or more of: CD25, CD69, HLA-DR CD8 Breast Carcinoma Depletion 5E11, BRST1 * Antibodies in standard negative selection cocktails are shown in blue. Antibodies in standard positive selection cocktails are shown in yellow. Antibodies suggested for addition to a standard cocktail for the enrichment of a particular cell subset are shown in red (custom cocktail).

6 Antibody Cocktails T cells Cocktail to Enrich Cell Type for labeling CD4 T cells NaÏve CD4 T cells CD8 T cells B cells NK cells Dendritic cells Hematopoietic Progenitors Primitive Hematopoietic Progenitors Monocytes T Cell Depletion Human 1 x 10 9 cells (2 ml) 5 x 10 8 cells (10 ml) * 14661* Human Positive 1 x 10 9 cells (1 ml) [2 x 10 9 cells - 1 ml] [14756] Mouse 5 x 10 8 cells (0.1 ml) 1 x 10 9 cells (0.2 ml) 5 x 10 9 cells (1 ml) Rat 5 x 10 8 cells (1 ml) 1 x 10 9 cells (2 ml) 5 x 10 9 cells (10 ml) Non-Human Primate 1 x 10 9 cells (2 ml) 5 x 10 9 cells (10 ml) Custom CD4 Depletion CD8 Depletion 14652* 14662* Breast Carcinoma Cell Depletion CML De-Bulking Human Cells from Mouse/Human Chimeras Basophils Eosinophils Granulocytes Epithelial Tumor Cells 14653* 14663* 14154* 14164* * 14161* StemCell Technologies In North America Tel: Fax: Toll Free Tel: Toll Free Fax: info@stemcell.com orders@stemcell.com options for all species anti-biotin tetrameric antibody complexes (tac) Catalog Numbers: ml for labeling 5 x 10 8 cells ml for labeling 1 x 10 9 cells ml for labeling 5 x 10 9 cells anti-r-phycoerythrin (pe) tetrameric antibody complexes (tac) Catalog Numbers: ml for labeling 5 x 10 8 cells ml for labeling 1 x 10 9 cells ml for labeling 5 x 10 9 cells In the United Kingdom Tel: 44.(0) Fax: 44.(0) Toll Free within United Kingdom: Tel: Fax: info@stemcellgb.com In Europe Tel: 33.(0) Fax: 33.(0) info@stemcellfrance.com Columns (Supplied in 10 column packs with the exception of the 1.0 column which is sold individually) Catalog Number Gravity Pump Column Diameter Optimum Cell Number per Column** Will Fit Magnet Size x 10 6 cells red, green x 10 7 cells all sizes x 10 8 cells green, blue, black x 10 8 cells green, blue, black x cells black **When using Ficolled cells, optimum cell number per column can be doubled. Note: Not to be used for positive selection. Magnets and Other Equipment Catalog Number Description For a complete list of StemSep support products, please see our catalog or website < TM Unlike other immunomagnetic cell separation systems, StemSep does not require coupling the anti-cell antibodies directly to magnetic particles. Instead, StemSep utilizes flexible TAC technology. The colloidal paramagnetic nanoparticles used in StemSep are coated with dextran, allowing them to be bound by the anti-dextran end of any StemSep TAC reagent. Since the anti-cell end of a bi-specific TAC reagent can be varied, TAC can be easily customized by incorporating any cell-specific mouse IgG 1 monoclonal antibody, instantly creating a novel cell separation reagent. If a mouse IgG 1 monoclonal is not available for a particular cell surface antigen, we provide anti-biotin or anti-pe TAC, allowing the use of any biotinylated or PE-conjugated cell-specific antibodies in your separations. This flexibility of StemSep and our custom cocktail program have allowed many researchers to perform unique cell separations that would otherwise not have been possible. Tetrameric Antibody Complexes Pump or Gravity Feed Number of Simultaneous Separations red magnet gravity and stand or pump 0.3 Advantage: TAC Flexibility Column Size green magnet gravity 1 0.1, 0.3 and stand or pump 0.5, blue magnet pump 1 to 4 0.3, 0.5 and stand black magnet pump 1 0.3, 0.5 and stand 0.6", peristaltic - 1 to 4 0.1, 0.3, pump 0.5, *This product is available only as a custom order. Please contact us for more information. Please contact us directly or one of our international distributors at: Australia, ; Austria, ; Belgium, ; Czech Republic, ; Denmark, ; Finland, ; France, 33.(0) ; Germany, ; Greece, ; Iceland, ; India, ; Ireland, 44.(0) ; Israel, ; Italy, ; Japan, ; Korea, ; Luxembourg, ; Netherlands, ; New Zealand, Norway, ; Poland, ; Portugal, ; P.R. China, ; Singapore, ; Slovenia, ; South Africa, 27.(0) ; Spain, ; Sweden, ; Switzerland, ; Taiwan, ; Turkey, ; United Kingdom, 44.(0)

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