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Positive Cell Isolation Low impact on cells, high impact on results Dynabeads® FlowComp™ technology for positive isolation of human and mouse cells 105 105 104 103 102 104 102 103 104 105 103 102 102 103 Positive Cell Isolation Low impact on cells, high impact on results Dynabeads FlowComp™ technology ® →→ Isolate from whole blood, buffy coat, or mononuclear cells Isolate bead-free cells →→ Release the cells and remove the beads In contrast to column-based systems, the Dynabeads® FlowComp™ →→ Better purity and viability technology allows you to isolate bead-free cells directly from human →→ Tube-based method, no columns required whole blood, buffy coat, or from mouse/human mononuclear cells (MNC) (Figure 1). Following separation, the beads are immediately The immune system protects us from harmful substances released and removed, ensuring the least possible interaction with through a complex interplay of immune cells. Cell isolation is the your cells. first of many steps in immunological workflows, and exposure of your cells to certain foreign substances during this step can influ- Column-free method ence your results. With the tube-based Dynabeads® technology, your cells are not exposed to the stress of being passed through a dense column. Avoid artifacts Dynabeads® are larger and have a higher iron content, allowing When using column-based systems with “biodegradable” nano- for faster kinetics and better separation. The isolated bead-free particles, the magnetic particles are left on the cells after positive cells maintain their functional characteristics (Figure 2) and are isolation. There is growing concern among scientists about what ready for flow cytometric analysis or any downstream cell-based the short- and long-term effects of the continued exposure of assay [3–7]. Dynabeads® are very stable (shelf life >2 years), are not cells to iron oxides, sugar (dextran), and other potentially harmful biodegradable, and show no leakage of iron or other immuno- substances may be [1–2]. gens that could potentially influence your results. Dynabeads® bind to target cells during incubation. Separate beads and cells using the magnet. Bead Modified streptavidin Start with human whole blood/buffy coat or MNC from mouse/human samples. Incubate with provided antibody mix and add Dynabeads®. Apply Dynal® magnet to separate bead-bound cells. Discard supernatant. Add FlowComp™ buffer to release target cells from Dynabeads®. Modified biotin Cell Release buffer Isolated bead-free cells are ready for use in any downstream application, including flow cytometry. Figure 1. Overview of the simple isolation procedure. The starting sample is mouse/human mononuclear cells (MNC) or human whole blood/buffy coat. 2 Before isolation 105 Propidium iodide 100 Count 75 Better purity and viability 50 25 0 For easier flow cytometer read-out, some researchers choose to 104 103 102 102 103 104 105 102 CD4+ 103 104 105 CD4+ only gate for the live cells when analyzing their cells. Thus, the Column-based isolation of mouse CD4+ T cells apoptotic or dead cells are excluded from the analysis, but not Purity: 78.5% 400 from the sample itself. Dynabeads® FlowComp™ Mouse CD4 yield 105 Propidium iodide 350 cells), compared to as little as 49% recovery with a column-based Count 300 83% total recovery (total number of live, non-apoptotic CD4 + 250 200 150 100 method (Figure 3). With Dynabeads® FlowComp™ technology, 104 103 102 Viability: 63% 50 0 cell purity is also consistently superior (Figure 4). 10 10 2 3 10 4 10 10 2 5 10 3 10 105 4 CD4+ CD4+ Dynabeads® FlowComp™ isolation of mouse CD4+ T cells 600 Purity: 97.2% Freshly isolated Day 0 400 Count 105 105 Propidium iodide 500 105 300 CD14 APC-A 104 103 102 104 0 103 103 104 105 102 CD86 PE-A 103 104 105 HLA DR FITC-A Day 6 CD14 APC-A 104 103 102 103 104 CD86 PE-A 105 10 4 10 5 Viability: 86% 102 103 104 105 CD4+ 100 104 80 103 102 102 10 3 Figure 3. Isolation of CD4+ T cells from mouse spleen cells. Cell isolation using Dynabeads® FlowComp™ Mouse CD4 results in substantially higher purity (97%) and viability (86%) than column-based positive cell isolation (yielding purity and viability of 78% and 63%, respectively). 105 105 CD14 APC-A 10 2 CD4+ 102 102 103 102 100 102 103 104 105 HLA DR FITC-A Figure 2. Isolated cells keep their functional characteristics. Dynabeads® FlowComp™ Mouse CD14 was used to isolate monocytes from whole blood. After 6 days of culturing with IL-4 and GM-CSF, the cells differentiated into monocyte-derived dendritic cells (Mo-DC). As seen, CD14 was lost during culture and cells up-regulated HLA-DR and CD86. More than 60% recovery of Mo-DC was observed. Similar results were observed when starting with MNC or buffy coat. Purity (%) CD14 APC-A 200 104 60 40 20 Dynabeads® FlowComp™ Human CD8 (n = 63) Column-based isolation (n = 5) 0 Figure 4. Isolation of CD8+ T cells from peripheral blood mononuclear cells. Isolation with Dynabeads® FlowComp™ Human CD8 gave high purity and minimal performance variations. 3 www.invitrogen.com ”Love it; looks like over 90% recovery for the Dynabeads® FlowComp™ Mouse CD4 kit, even from raw splenocytes. I definitely intend to use it.” Bindu Kanathezhath, MD Children’s Hospital and Research Center, Oakland, California USA To learn more about the high recovery, viability, and purity you can get with Dynabeads® FlowComp™ technology, visit www.invitrogen.com/flowcomp. Ordering information Product Amount processed Cat. No. Dynabeads® FlowComp™ Human CD4 80 mL whole blood/buffy coat*/2 x 109 cells 113-61D Dynabeads® FlowComp™ Human CD8 80 mL whole blood/buffy coat*/2 x 109 cells 113-62D Dynabeads® FlowComp™ Human CD3 80 mL whole blood/buffy coat*/2 x 109 cells Positive isolation of human cells 113-65D 9 Dynabeads® FlowComp™ Human CD14 80 mL whole blood/160 mL buffy coat/2 x 10 cells 113-67D Dynabeads® FlowComp™ Human CD45RA 2 x 109 cells 113-68D Dynabeads® FlowComp™ Mouse CD4 2 x 109 cells 114-61D Dynabeads® FlowComp™ Mouse CD8 2 x 109 cells 114-62D Dynabeads® FlowComp™ Mouse Pan T (CD90.2) 9 2 x 10 cells 114-65D Dynabeads® FlowComp™ Mouse CD4+CD25+ Treg Cells 1 x 109 cells 114-63D Dynabeads® FlowComp™ Mouse CD49b 2 x 109 cells 114-64D Dynabeads® FlowComp™ Flexi 2 x 109 cells 110-61D DynaMag™ magnets See www.invitrogen.com/magnets for magnet recommendations. HulaMixer™ Sample Mixer 1 unit, holds 0.5 mL–50 mL tubes Positive isolation of mouse cells Related products 159-20D * For isolation from buffy coat, contact Technical Support for specific protocols. For current prices, please visit www.invitrogen.com. Visit www.invitrogen.com/immunology for relevant antibodies, cell expansion technology, and flow cytometry reagents. References 1. Pisanic TR II et al. (2007) Nanotoxicity of iron oxide nanoparticle internalization in growing neurons. Biomaterials 28:2572–2581. 2. Berry, CC et al. (2004) Cell response to dextran-derivatised iron oxide nanoparticles post internalisation. Biomaterials 25:5405–5413. 3. Feng X et al. (2010) Foxp1 is an essential transcriptional regulator for the generation of quiescent naive T cells during thymocyte development. Blood 115(3):510–518. 4. Del Cacho E et al. (2009) Avian follicular and interdigitating dendritic cells: Isolation and morphologic, phenotypic, and functional analyses. Vet Immunol Immunopathol 129:66–75. 5. Wang J et al. (2009) De novo generation and enhanced suppression of human CD4+CD25+ regulatory T cells by retinoic acid. J Immunol 183:4119–4126. 6. Brody JD et al. (2009) Immunotransplantation preferentially expands T-effector cells over T-regulatory cells and cures large lymphoma tumors. Blood 113(1):85–94. 7. Huber SA (2009) Depletion of γδ+ T cells increases CD4+ FoxP3 (T regulatory) cell response in coxsackievirus B3-induced myocarditis. Immunology 127:567–576. DYNAL® has pioneered magnetic separation technologies for biological discovery that are both simple and highly reproducible. Based on their patented superparamagnetic, monodisperse beads, Dynabeads® technologies represent a superior paradigm for cell and biomolecule separation in a wide range of basic and clinical research applications, diagnostic assays, and therapeutic protocols. www.invitrogen.com For research use only. Not intended for any animal or human therapeutic or diagnostic use, unless otherwise stated. © 2010 Life Technologies Corporation. All rights reserved. The trademarks mentioned herein are the property of Life Technologies Corporation or their respective owners. These products may be covered by one or more Limited Use Label Licenses (see Invitrogen catalog or www.invitrogen.com). By use of these products you accept the terms and conditions of all applicable Limited Use Label Licenses. CO21311 0510