BD IMag Cell Separation System

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1 BD IMag Cell Separation System

2 Table of Contents Magnetic Cell Separation with BD IMag Particles BD IMag Enrichment Protocol Flow Chart BD IMag Enrichment Reagents Negative Selection versus Positive Selection Sample Data The Flexibility of the BD IMag Cell Separation System Sample Data BD IMag Sets for Enrichment BD IMag Reagents for Positive Selection For Research Use Only. Not for use in diagnostic or therapeutic procedures. All applications are either tested in-house or reported in the literature. See Technical Data Sheets for details. BD flow cytometers are class I () laser products. Purchase does not include or carry any right to resell or transfer this product either as a stand-alone product or as a component of another product. Any use of this product other than the permitted use without the express written authorization of Becton Dickinson and Company is strictly prohibited. BD, BD Logo and all other trademarks are the property of Becton, Dickinson and Company BD 2

3 Magnetic Cell Separation with BD IMag Particles The BD IMag Cell Separation System is based on a simple yet highly effective direct magnet technology that allows for the rapid positive and/or negative selection (enrichment) of cell populations without the use of magnetic separation columns. When using the BD IMag system, one can expect excellent purities and recoveries of specific leukocyte subpopulations in a few short steps. BD IMag particles are nanometer-sized superparamagnetic particles with monoclonal antibody or streptavidin covalently bound to their surface. These particles have been optimized for both positive and negative selection of leukocyte sub-populations using the BD IMagnet direct magnet (DM) system (figure ). Positive selections typically make use of BD IMag particles that have monoclonal antibodies covalently bound to their surface. These particles specifically target a leukocyte sub-population of interest. Once placed within the BD IMagnet, targeted cells migrate toward the magnet and are retained within the magnetic field while the unlabeled cells are drawn off. The targeted cells can then be collected and used in the desired application ater removal from the magnetic field. In the event that negative selection (enrichment) is required, the unlabeled cells are drawn off and can be utilized for a variety of applications such as cell sorting. Figure. BD IMagnet direct magnet system

4 BD IMag Enrichment Protocol Flow Chart BD IMag labeled cell suspension Enriched (depleted) fraction Ready for analysis or culture 2 - BD IMagnet: 6-8 minutes Positive fraction Resuspend positive fraction BD IMagnet: 6-8 minutes (optional) BD IMagnet: 6-8 minutes Discard residual positive fraction Positive fraction Resuspend positive fraction BD IMagnet: 6-8 minutes Positive fraction Ready for Analysis or Culture 6 + Final enriched fraction Ready for Analysis or Culture Figure 2. BD IMag Flexible Enrichment Protocol Place the BD IMag labeled cell suspension onto the BD IMagnet. 2 - Remove the supernatant containing the negative fraction and place in an appropriately labeled tube. 2 + Remove the tube containing the positive fraction. 3 Resuspend the positive fraction and place back onto the BD IMagnet. 4 - Remove the negative fraction and pool with the negative fraction from step Remove the tube containing the positive fraction. 5 Resuspend the positive fraction and place back onto the BD IMagnet. 6 - Remove the negative fraction and pool with the negative fraction from steps 2 - and Remove the positive fraction and resuspend the cells for use. 7 Place the combined negative fractions on the BD IMagnet. 8 - Remove remaining twice enriched fraction and place in an appropriately labeled tube for use. 8 + Remove and discard residual positive fraction. 4

5 BD IMag Enrichment Reagents Enrichment by depletion or negative selection is used for research applications which require a cell population with high levels of purity and no antibody or particles bound to their surface. In this instance, all unwanted cells are first labeled with a cocktail containing monoclonal antibodies against antigens expressed by all unwanted cells. After washing away unbound antibody, a second-step BD IMag reagent is used to magnetically label these cells. The labeled cells migrate to the BD IMagnet leaving behind a pure and untouched sub-population of cells to be drawn off (figure 2). BD Biosciences Pharmingen has significantly expanded the BD IMag product line. In addition to the human and mouse positive selection reagents, there are now human and mouse enrichment sets providing researchers with a cost-effective method for isolating untouched target cells with exceptionally high levels of purity and recovery (figures 3 and 4). Finally, there are several second-step BD IMag particle reagents that allow researchers to customize their magnetic cell separations using their own specific antibodies or antibody cocktails. BD IMag Competitor Target Cell Purity BD IMag Human Enrichment Sets vs. Competitor Target Cell Recovery BD IMag Human Enrichment Sets vs. Competitor Purity (% of final enriched fraction) Recovery (% of original cell population) B cells T cells CD4 T cells CD8 T cells NK cells 0 B cells T cells CD4 T cells CD8 T cells NK cells Figure 3a. A comparison of human target cell purity obtained after enrichment of different lymphocyte sub-populations from PBMC using either the BD IMag Human Enrichment Sets or the competitor s corresponding magnetic separation column products. Figure 3b. A comparison of human target cell recovery obtained after enrichment of different lymphocyte sub-populations from PBMC using either the BD IMag Human Enrichment Sets or the competitor s corresponding magnetic separation column products. Target Cell Purity BD IMag Mouse Enrichment Sets vs. Competitor Target Cell Recovery BD IMag Mouse Enrichment Sets vs. Competitor Purity (% of final enriched fraction) Recovery (% of original cell population) B cells T cells CD4 T cells CD8 T cells NK cells Hematopoietic Progenitor cells Dendritic Cells* 0 B cells T cells CD4 T cells CD8 T cells NK cells Hematopoietic Dendritic Progenitor cells Cells* Figure 4a. A comparison of mouse target cell purity obtained after enrichment of different lymphocyte sub-populations from spleen (or bone marrow for hematopoietic progenitors) using either the BD IMag Mouse Enrichment Sets or the competitor s corresponding magnetic separation column products. Figure 4b. A comparison of mouse target cell recovery obtained after enrichment of different lymphocyte sub-populations from spleen (or bone marrow for hematopoietic progenitors) using either the BD IMag Mouse Enrichment Sets or the competitor s corresponding magnetic separation column products. * Comparison not available 5

6 + Negative Selection versus Positive Selection Positive cell selections yield excellent results with respect to purity, recovery, and viability of selected cells. However, depending on the cell type being selected and the surface antigen being targeted by the particle, positive selections can result in cells becoming activated or otherwise functionally altered. Even though the probability of activation is low, this magnetic particle-induced activation may be an issue for researchers who specifically require purified yet unstimulated cells. As such, they should consider negative selection for their cell separations. Negative selection is a simple enrichment process by which unwanted cells are magnetically labeled and removed leaving an untouched population of target cells. To demonstrate how little magnetic particle-induced activation occurs with negative selection, mouse CD4 T cells were isolated either by positive or negative selection and then placed in culture for 48 hours, at which time the surface expression of activation markers CD25 and CD69 were examined (figure 5). CD4 T cells were either positively selected from mouse spleen using BD IMag Anti- Mouse CD4 Particles DM or enriched using the BD IMag Mouse CD4 T Lymphocyte Enrichment Set DM. For flow cytometric analysis of the two separation techniques, unmanipulated BALB/c splenocytes (figure 5A), the positively selected CD4 T cells (figure 5B), and the enriched (negatively selected) CD4 T cells (figure 5C) were stained with APC anti-cd3e (clone 45-2C) and PE anti- CD4 (clone GK.5). The percent CD3+/CD4+ cells in each sample is given. Immediately after isolation, the purified CD4 T cells were either placed in uncoated wells containing normal culture media or they were suspended in wells pre-coated with anti-cd3 (clone 7A2) and containing media with soluble anti- CD28 (clone 37.5). After 48 hours in culture, the cells were stained with PE anti-cd25 (PC6) and FITC anti-cd69 (H.2F3) to detect activated lymphocytes, and propidium iodide to detect dead cells. Positively selected cells that had been suspended in media alone had more dead cells when compared to the enriched fraction (compare figures 5D and 5F) and of the viable cells, approximately 28% were CD25+ (figure 5E). In contrast, the enriched cells had approximately 5% CD25+ cells (figure 5G). In either case, very little CD69 expression was seen. When comparing the cells that had been cultured in the presence of anti-cd3 and anti-cd28, enriched cells still had a greater viability after 48 hours (compare figures 5H and 5J), but there was virtually no difference in the expression pattern of CD25 and CD69 (compare figures 5I and 5K). 6

7 00 A: Unseparated splenocytes 00 B: Positively selected CD4 T cells 00 C: Enriched (negatively selected) CD4 T cells PE CD4 9.8 PE CD PE CD APC CD APC CD APC CD3 Media Alone D: Positive selection media alone 00 F: Enriched media alone 00 Propidium Iodine 0 0 Propidium Iodine Forward Scatter Forward Scatter E: Positive selection media alone G: Enriched media alone PE CD25 PE CD FITC CD FITC CD69 Activated H: Positive selection activated 00 J: Enriched activated Propidium Iodine Propidium Iodine Forward Scatter Forward Scatter I: Positive selection activated K: Enriched activated PE CD25 PE CD FITC CD FITC CD69 Figure 5. Expression of activation markers CD25 and CD69 after either positive selection or negative selection (enrichment) of CD4 T cells using the BD IMag Mouse CD4 Particles DM and BD IMag Mouse CD4 T Lymphocyte Enrichment Set DM respectively. 7

8 The Flexibility of the BD IMag Cell Separation System While the basic enrichment (negative selection) protocol (figure 2) has been optimized to maximize both purity and recovery, the BD IMag system has sufficient flexibility to allow any separation to be customized for a particular researcher s needs. For example, by reducing the recommended number of washes, even greater purity can be obtained. Alternately, by increasing the number of washes and/or omitting the final incubation on the BD IMagnet (figure 2, step 7) the recovery can be increased. Another advantage of negative selections is the ability to do a positive selection using the enriched cells as a starting material. This makes the isolation of uncommon cell sub-populations possible in just a few simple steps. The examples below demonstrate how the simple BD IMag enrichment protocol can be manipulated to increase purity and isolate specific cell sub-populations within the CD4 T cell subset. Maximizing the Purity of Enriched Cells CD4 T cells were isolated from human peripheral blood mononuclear cells (PBMC) using the BD IMag Human CD4 T Lymphocyte Enrichment Set DM. For flow cytometric analysis, the unmanipulated PBMCs (figure 6A), the combined once enriched fraction (figure 6B), and the twice enriched fraction (figure 6C), were stained with APC anti- CD4 (RPA-T4) to detect CD4 T cells and a mixture of PE anti-cd8 (RPA-T8), CDb (ICRF44), CD6 (3G8), CD9 (HIB9), CD36 (CB38), CD56 (B59), CD23 (7G3), glycophorin A (GA-R2), and γ/δ TCR (B) to detect non-cd4 T leukocytes and erythrocytes. The percent CD4 T cells in each sample is given. The once-enriched CD4 T cell fraction was obtained after 3 incubations on the BD IMagnet (steps through 6-, figure 2). To increase purity, this enriched fraction was placed on the BD IMagnet a final time to give a twice-enriched fraction (steps 7 through 8-, figure 2). In general, this additional incubation will result in a 2% 20% increase in purity but may reduce recovery by up to 0%. Factors that can influence purity and recovery include the anti-coagulant used, the quality of the sample preparation, the donor, the specific cell type being enriched, and the percent target cells in the unmanipulated sample. In some instances, an even purer sample of CD4 T cells is required. For example, when purifying T regulatory cells which are CD4+/CD25+ it is preferred to have no contaminating non-cd4 T cells present. In such cases we recommend eliminating all washes (no steps 3 and 5, figure 2) before obtaining the twiceenriched fraction. For the CD4 T cell enrichment, one can then expect purities greater than 98%. However, recoveries may be up to 30% lower. In the example shown here, CD4 T cells were first enriched from PBMC using the BD IMag Human CD4 T Lymphocyte Enrichment Set DM, followed by a CD25 positive selection using the BD IMag Anti-Human CD25 particles - DM. Unmanipulated PBMC (figure 6D), the CD4 enriched fraction (figure 6E, prepared with no wash steps), and the T regulatory CD4+/CD25+ enriched fraction (figure 6F), were stained with PE anti-cd4 (RPA- T4) and APC anti-cd25 (M-A25). The percent total CD4+ cells and the percent CD4+/CD25+ T regulatory cells in each sample are given. Subpopulation Selection from Enriched Cells CD4 T cells can also be divided into naïve cells that have not seen antigen and express CD45RA and those cells that have seen antigen (activated or memory) and express CD45RO. Both of these CD4 T cell subsets can be isolated to high levels of purity by first doing a CD4 enrichment followed by a CD45RO positive selection. Since CD45RO and CD45RA are almost mutually exclusive on CD4 T cells, the CD45RO-negative fraction would be expected to contain the CD45RA+ expressing cells. Again, CD4 T cells were first enriched from PBMC using the BD IMag Human CD4 T Lymphocyte Enrichment Set DM, followed by a CD45RO positive selection using the BD IMag Anti-Human CD45RO Particles DM. For flow cytometric analysis, enriched CD4 T cells (figure 6G), the CD45RO positive fraction (figure 6H) and the CD45RO negative fraction (figure 6I) were stained with APC anit-cd4 (RPA-T4) and FITC anti-cd45ro (UCHL). The percent CD4+/CD45RO+ cells in each sample are given. To show that the CD45RO negative fraction contained the CD45RA+ cells, this fraction was also stained with APC anti-cd4 (RPA-T4) and FITC anti- CD45RA (HI) (figure 6J). The percent CD4+/CD45RA+ cells is given. Therefore, by combining the use of the CD4 T cell enrichment set with the CD45RO positive selection reagent, it was possible to isolate highly purified populations of CD4+/CD45RO+ and CD4+/CD45RA+ cells in just a few simple steps. 8

9 00 A: Unmanipulated PBMC 00 B: Combined Enriched Fraction 00 C: Twice Enriched Fraction PE Cocktail PE Cocktail PE Cocktail D: Unmanipulated PBMC 00 E: Enriched CD4 T cells 00 F: CD4+/CD25+ T regulatory cells PE CD4 PE CD4 PE CD APC CD APC CD APC CD25 G: Enriched CD4 T cells 00 H: CD45RO-Positive Fraction 00 I: CD45RO-Negative Fraction 00 J: CD45RO-Negative Fraction FITC CD45R FITC CD45R FITC CD45R FITC CD45RA Figure 6. Demonstration of how the basic enrichment protocol can be manipulated for different experimental needs and how positive selections can be coupled with enrichments to isolate uncommon cell subpopulations. 9

10 BD IMag Sets for Enrichment BD IMag Sets for Enrichment Human DESCRIPTION REACT CONTENTS APPS FORMAT SIZE CAT. NO. B Lymphocyte Enrichment Set - DM Hu Biotinylated antibody cocktail and BD IMag Streptavidin Particles Sep BD IMag-DM x 0 9 Cells May 2004 CD4 T Lymphocyte Enrichment Set - DM Hu Biotinylated antibody cocktail and BD IMag Streptavidin Particles Sep BD IMag-DM x 0 9 Cells CD8 T Lymphocyte Enrichment Set - DM Hu Biotinylated antibody cocktail and BD IMag Streptavidin Particles Sep BD IMag-DM x 0 9 Cells NK Cell Enrichment Set - DM Hu Biotinylated antibody cocktail and BD IMag Streptavidin Particles Sep BD IMag-DM x 0 9 Cells May 2004 T Lymphocyte Enrichment Set - DM Hu Biotinylated antibody cocktail and BD IMag Streptavidin Particles Sep BD IMag-DM x 0 9 Cells Mouse DESCRIPTION REACT CONTENTS APPS FORMAT SIZE CAT. NO. B Lymphocyte Enrichment Set - DM Ms Biotinylated antibody cocktail and BD IMag Streptavidin Particles Sep BD IMag-DM x 0 9 Cells CD4 T Lymphocyte Enrichment Set - DM Ms Biotinylated antibody cocktail and BD IMag Streptavidin Particles Sep BD IMag-DM x 0 9 Cells 5583 CD8 T Lymphocyte Enrichment Set - DM Ms Biotinylated antibody cocktail and BD IMag Streptavidin Particles Sep BD IMag-DM x 0 9 Cells Dendritic Cell Enrichment Set - DM Ms Biotinylated antibody cocktail and BD IMag Streptavidin Particles Sep BD IMag-DM x 0 9 Cells Hematopoietic Progenitor Cell Ms Biotinylated antibody cocktail and BD IMag Streptavidin Particles Sep BD IMag-DM x 0 9 Cells Enrichment Set - DM NK Cell Enrichment Set - DM Ms Biotinylated antibody cocktail and BD IMag Streptavidin Particles Sep BD IMag-DM x 0 9 Cells T Lymphocyte Enrichment Set - DM Ms Biotinylated antibody cocktail and BD IMag Streptavidin Particles Sep BD IMag-DM x 0 9 Cells DM - for use with the BD IMagnet Direct Magnet MSC - for use with Magnetic Separation Columns BD IMag particles are prepared from Carboxy-functionalized magnetic particles manufactured by Skold Technology 0

11 BD IMag Reagents for Positive Selection Human DESCRIPTION REACT CLONE APPS FORMAT SIZE CAT. NO. CD3 Magnetic Particles - DM Hu HIT3a Sep BD IMag-DM x 0 9 Cells CD3 Magnetic Particles - MSC Hu HIT3a Sep BD IMag-MSC x 0 9 Cells CD4 Magnetic Particles - DM Hu, Rhe L200 Sep BD IMag-DM x 0 9 Cells CD4 Magnetic Particles - MSC Hu, Rhe L200 Sep BD IMag-MSC x 0 9 Cells CD8 Magnetic Particles - DM Hu, Rhe SK Sep BD IMag-DM x 0 9 Cells CD8 Magnetic Particles - MSC Hu, Rhe SK Sep BD IMag-MSC x 0 9 Cells CD4 Magnetic Particles - DM Hu, Rhe MφP9 Sep BD IMag-DM x 0 9 Cells CD4 Magnetic Particles - MSC Hu, Rhe MφP9 Sep BD IMag-MSC x 0 9 Cells CD9 Magnetic Particles - DM Hu HIB9 Sep BD IMag-DM x 0 9 Cells CD9 Magnetic Particles - MSC Hu HIB9 Sep BD IMag-MSC x 0 9 Cells 5552 CD25 Magnetic Particles - DM Hu 2A3 Sep BD IMag-DM x 0 9 Cells CD45RA Magnetic Particles - DM Hu HI Sep BD IMag-DM x 0 9 Cells CD45RO Magnetic Particles - DM Hu UCHL Sep BD IMag-DM x 0 9 Cells CD56 Magnetic Particles - DM Hu, Rhe NCAM6.2 Sep BD IMag-DM x 0 9 Cells γ/δ T Cell Receptor Separation Set - DM Hu B Sep BD IMag-DM x 0 9 Cells May 2004 Mouse DESCRIPTION REACT CLONE APPS FORMAT SIZE CAT. NO. CD4 Magnetic Particles - DM Ms GK.5 Sep BD IMag-DM 2 x 0 9 cells CD4 Magnetic Particles - MSC Ms GK.5 Sep BD IMag-MSC 2 x 0 9 cells CD8a Magnetic Particles - DM Ms Sep BD IMag-DM 2 x 0 9 cells 5556 CDb Magnetic Particles - DM Ms M/70 Sep BD IMag-DM 2 x 0 9 cells CDb Magnetic Particles - MSC Ms M/70 Sep BD IMag-MSC 2 x 0 9 cells 5580 CD45R/B220 Magnetic Particles - DM Ms RA3-6B2 Sep BD IMag-DM 2 x 0 9 cells 5553 CD90.2 (Thy.2) Magnetic Particles - DM Ms 30-H2 Sep BD IMag-DM 2 x 0 9 cells 5558 Ly-6G and Ly-6C (Gr-) Magnetic Particles - DM Ms RB6-8C5 Sep BD IMag-DM 2 x 0 9 cells 558 Ly-6G and Ly-6C (Gr-) Magnetic Particles - MSC Ms RB6-8C5 Sep BD IMag-MSC 2 x 0 9 cells NK Cell Separation Set - DM Ms DX5 Sep BD IMag-DM 2 x 0 9 cells May 2004 Other DESCRIPTION REACT CLONE APPS FORMAT SIZE CAT. NO. Allophycocyanin (APC) Magnetic Particles - DM E30-22 Sep BD IMag-DM x 0 9 cells R-Phycoerythrin (PE) Magnetic Particles - DM E3-459 Sep BD IMag-DM x 0 9 cells Mouse IgG Magnetic Particles - DM A85- Sep BD IMag-DM x 0 9 cells Rat Ig, κ light chain Magnetic Particles - DM MRK- Sep BD IMag-DM x 0 9 cells Streptavidin Magnetic Particles Plus - DM Sep BD IMag-DM x 0 9 cells Streptavidin Magnetic Particles Plus - MSC Sep BD IMag-MSC x 0 9 cells 5578 Buffer (0x) for use with BD IMag cell separation products Sep Buffer ml BD IMagnet Cell Separation Magnet Sep Magnet each 5523 DM - for use with the BD IMagnet Direct Magnet MSC - for use with Magnetic Separation Columns BD IMag particles are prepared from Carboxy-functionalized magnetic particles manufactured by Skold Technology Visit for additional product details.

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