BD IMag Cell Separation System

BD IMag Cell Separation System

Table of Contents Magnetic Cell Separation with BD IMag Particles.............................................................. 3 BD IMag Enrichment Protocol Flow Chart...................................................................... 4 BD IMag Enrichment Reagents............................................................................... 5 Negative Selection versus Positive Selection..................................................................... 6 Sample Data........................................................................................... 7 The Flexibility of the BD IMag Cell Separation System............................................................ 8 Sample Data........................................................................................... 9 BD IMag Sets for Enrichment................................................................................ 0 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. 2004 BD 2 www.bdbiosciences.com

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

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 7 4-2 + Positive fraction Resuspend positive fraction 3 6 - BD IMagnet: 6-8 minutes (optional) BD IMagnet: 6-8 minutes Discard residual positive fraction Positive fraction 5 8 + 8-4 + 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 2 -. 4 + 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 4 -. 6 + 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 www.bdbiosciences.com

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) 80 60 40 20 Recovery (% of original cell population) 80 60 40 20 0 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) 80 60 40 20 Recovery (% of original cell population) 80 60 40 20 0 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

+ 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 www.bdbiosciences.com

00 A: Unseparated splenocytes 00 B: Positively selected CD4 T cells 00 C: Enriched (negatively selected) CD4 T cells 0 0 0 PE CD4 9.8 PE CD4 95.4 PE CD4 93.9 0 0 0 0 0 00 APC CD3 0 0 00 APC CD3 0 0 00 APC CD3 Media Alone D: Positive selection media alone 00 F: Enriched media alone 00 Propidium Iodine 0 0 Propidium Iodine 0 0 58.9 0 50 50 200 250 Forward Scatter 88.9 0 50 50 200 250 Forward Scatter E: Positive selection media alone 00 24.6 3.5 G: Enriched media alone 00 4.4 0.9 0 0 PE CD25 PE CD25 0 0 68.4 7.6 0.3 0 0 00 FITC CD69 93.9 94.5 0.2 0 0 00 FITC CD69 Activated H: Positive selection activated 00 J: Enriched activated 00 0 0 Propidium Iodine Propidium Iodine 0 0 58.9 0 50 50 200 250 Forward Scatter 72.2 0 50 50 200 250 Forward Scatter I: Positive selection activated 00 0.9 98.9 K: Enriched activated 00.8 96.5 0 0 PE CD25 PE CD25 0 0 93.9 0. 0. 0 0 00 FITC CD69 93.9.2 0.5 0 0 00 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

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 www.bdbiosciences.com

00 A: Unmanipulated PBMC 00 B: Combined Enriched Fraction 00 C: Twice Enriched Fraction 0 0 0 PE Cocktail PE Cocktail PE Cocktail 0 0 0 23.7 0 0 00 84.7 0 0 00 93.6 0 0 00 00 D: Unmanipulated PBMC 00 E: Enriched CD4 T cells 00 F: CD4+/CD25+ T regulatory cells 40.3 6. 98.8 43. 99. 88.9 0 0 0 PE CD4 PE CD4 PE CD4 0 0 0 0 0 00 APC CD25 0 0 00 APC CD25 0 0 00 APC CD25 G: Enriched CD4 T cells 00 H: CD45RO-Positive Fraction 00 I: CD45RO-Negative Fraction 00 J: CD45RO-Negative Fraction 00 0 0 0 0 49.4 90.8 3.2 98.5 0 0 0 0 0 0 00 FITC CD45R0 0 0 00 FITC CD45R0 0 0 00 FITC CD45R0 0 0 00 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

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 557939 CD8 T Lymphocyte Enrichment Set - DM Hu Biotinylated antibody cocktail and BD IMag Streptavidin Particles Sep BD IMag-DM x 0 9 Cells 55794 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 557874 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 557792 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 55847 Dendritic Cell Enrichment Set - DM Ms Biotinylated antibody cocktail and BD IMag Streptavidin Particles Sep BD IMag-DM x 0 9 Cells 557955 Hematopoietic Progenitor Cell Ms Biotinylated antibody cocktail and BD IMag Streptavidin Particles Sep BD IMag-DM x 0 9 Cells 55845 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 557954 T Lymphocyte Enrichment Set - DM Ms Biotinylated antibody cocktail and BD IMag Streptavidin Particles Sep BD IMag-DM x 0 9 Cells 557793 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 www.bdbiosciences.com

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 552593 CD3 Magnetic Particles - MSC Hu HIT3a Sep BD IMag-MSC x 0 9 Cells 552594 CD4 Magnetic Particles - DM Hu, Rhe L200 Sep BD IMag-DM x 0 9 Cells 557767 CD4 Magnetic Particles - MSC Hu, Rhe L200 Sep BD IMag-MSC x 0 9 Cells 557768 CD8 Magnetic Particles - DM Hu, Rhe SK Sep BD IMag-DM x 0 9 Cells 557766 CD8 Magnetic Particles - MSC Hu, Rhe SK Sep BD IMag-MSC x 0 9 Cells 557765 CD4 Magnetic Particles - DM Hu, Rhe MφP9 Sep BD IMag-DM x 0 9 Cells 557769 CD4 Magnetic Particles - MSC Hu, Rhe MφP9 Sep BD IMag-MSC x 0 9 Cells 557770 CD9 Magnetic Particles - DM Hu HIB9 Sep BD IMag-DM x 0 9 Cells 55520 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 558005 CD45RA Magnetic Particles - DM Hu HI Sep BD IMag-DM x 0 9 Cells 55798 CD45RO Magnetic Particles - DM Hu UCHL Sep BD IMag-DM x 0 9 Cells 557986 CD56 Magnetic Particles - DM Hu, Rhe NCAM6.2 Sep BD IMag-DM x 0 9 Cells 557775 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 55539 CD4 Magnetic Particles - MSC Ms GK.5 Sep BD IMag-MSC 2 x 0 9 cells 55540 CD8a Magnetic Particles - DM Ms 53-6.7 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 55803 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 55802 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 557932 R-Phycoerythrin (PE) Magnetic Particles - DM E3-459 Sep BD IMag-DM x 0 9 cells 557899 Mouse IgG Magnetic Particles - DM A85- Sep BD IMag-DM x 0 9 cells 557983 Rat Ig, κ light chain Magnetic Particles - DM MRK- Sep BD IMag-DM x 0 9 cells 558002 Streptavidin Magnetic Particles Plus - DM Sep BD IMag-DM x 0 9 cells 55782 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 552362 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 www.bdbiosciences.com/bdimag for additional product details.

Regional Offices Asia Pacific BD Singapore Tel 65.686.0633 Fax 65.6860.590 Australia/New Zealand Australia Tel 6.2.8875.7000 Fax 6.2.8875.7200 bd_anz@bd.com New Zealand Tel 64.9.574.2468 Fax 64.9.574.2469 bd_anz@bd.com Canada BD Biosciences Toll free 888.259.087 Tel 905.542.8028 Fax 888.229.998 canada@bd.com Europe Belgium Tel 32.53.720.2 Fax 32.53.720.450 contact_bdb@europe.bd.com Japan Nippon Becton Dickinson Toll free 020.8555.90 Tel 8.24.593.5405 Fax 8.24.593.576 Clontech Products Tel 8.3.5324.9609 Fax 8.3.5324.9637 United States BD Biosciences Customer/Technical Service Toll free 877.232.8995 Clontech Fax 650.354.0775 Discovery Labware Fax 978.90.7493 Immunocytometry Systems Fax 408.954.2347 Pharmingen Fax 858.82.8888 www.bdbiosciences.com Local Offices and Distributors Argentina/Paraguay/Uruguay Tel 54..455.7 x06 Fax 54..455.7400 Austria SCIENTIFIC SUPPORT Tel 43..706.36.60.44 Fax 43..706.36.60.45 BDBsupport_GSA@europe.bd.com Tel 43..706.36.60.0 Fax 43..706.36.60. Belgium Tel 32.53.720.600 Fax 32.53.720.220 scientific_support_benelux@europe.bd.com Tel 32.53.720.550 Fax 32.53.720.549 customer_service_bdbelgium@europe.bd.com Brazil Tel 55..585.9995 Fax 55..585.9895 biosciences@bd.com.br Central America/Caribbean Tel 506.290.738 Fax 506.290.733 Chile Tel 56.2 460.0380 x6 Fax 56.2 460.0306 China Tel 860.648.608 Fax 860.648.60 Colombia Tel 57..572.4060 x244 Fax 57..244.363 Denmark Tel 45.43.43.45.66 Fax 45.43.43.4.66 bdbsupport_dk@europe.bd.com East Africa Tel 254.2.3457 Fax 254.2.346 bd@africaonline.co.ke Eastern Europe Tel 49.622.305.6 Fax 49.622.305.48 bdb.ema@europe.bd.com Egypt Tel 202.268.08 Fax 202.266.7562 Finland Tel 358.9.88.70.7832 Fax 358.9.88.70.787 bdbsupport_fi@europe.bd.com France Tel 33.4.76.68.36.40 Fax 33.4.76.68.35.06 SCIENTIFIC SUPPORT Tel 33.4.76.68.34.25 Fax 33.4.76.68.55.7 bdb_france_scientific_support@europe.bd.com Tel 33.4.76.68.37.32 Fax 33.4.76.68.35.06 customerservice.bd.france@europe.bd.com Germany SCIENTIFIC SUPPORT Tel 49.622.305.525 Fax 49.622.305.530 BDBsupport_GSA@europe.bd.com Tel 49.622.305.55 Fax 49.622.303.609 customerservice.bdb.de@europe.bd.com Greece Tel 30.20.940.77.4 Fax 30.20.940.77.40 Hong Kong Tel 852.2575.8668 Fax 852.2803.5320 Hungary Szerena Tel 36..345.7090 Fax 36..345.7093 India Tel 9.24.238.3566.77 Fax 9.24.238.3225 Indonesia Tel 62.2.577.920 Fax 62.2.577.925 Italy Tel 39.02.48.240. Fax 39.02.48.20.33.36 Japan Fujisawa Pharmaceutical Co., Ltd. (Reagents from Immunocytometry Systems & Pharmingen) Tel 8.6.6206.7890 Fax 8.6.6206.7934 Korea Tel 822.3404.3700 Fax 822.557.4048 Malaysia Tel 603.7725.557 Fax 603.7725.4772 Mexico Tel 52.55.5999.8296 Fax 52.55.5999.8288 Middle East Tel 97.4.337.95.25 Fax 97.4.337.95.5 bdb.ema@europe.bd.com Netherlands Tel 3.20.582.94.24 Fax 3.20.582.94.26 scientific_support_benelux@europe.bd.com Tel 3.20.582.94.20 Fax 3.20.582.94.2 customer_service_bdholland@europe.bd.com North Africa Tel 33.4.76.68.35.03 Fax 33.4.76.68.35.44 bdbiosciences_maghreb@europe.bd.com Norway Immunocytometry Systems & Pharmingen Laborel S/A Tel 47.23.05.9.30 Fax 47.22.63.07.5 Peru/Bolivia/Ecuador Tel 5..430.0323 Fax 5..430.077 Philippines Tel 632.807.6073 Fax 632.850.998 Poland Tel 48.22.65.53.00 Fax 48.22.65.79.24 Portugal Enzifarma Tel 35.2.422.0.00 Fax 35.2.422.0.0 Saudi Arabia Tel 966..26.00.805/806 Fax 966..26.00.804 South Africa Tel 27..807.5 3 Fax 27..807.9 53 Spain Immunocytometry Systems & Pharmingen SCIENTIFIC SUPPORT Tel 34.9.848.8.77 Fax 34.9.848.8.05 Tel 34.902.27.7.27 Fax 34.9.848.8.04 Sweden Tel 46.8.775.5.0 Fax 46.8.775.5. bdbsupport_se@europe.bd.com Switzerland SCIENTIFIC SUPPORT Tel 4.6.485.22.9 Fax 4.6.485.22.92 BDBsupport_GSA@europe.bd.com Tel 4.6.485.22.22 Fax 4.6.485.22.00 customerservice.bdb.ch@europe.bd.com Taiwan Tel 8862.2722.5660 Fax 8862.2725.768 Thailand Tel 662.643.374 Fax 662.643.38 Turkey Tel 90.22.222.87.77 Fax 90.22.222.87.76 UK Tel 44.865.78.6.88 Fax 44.865.78.6.27 bduk_customer_service@europe.bd.com SCIENTIFIC SUPPORT bduk.sci.support@europe.bd.com Venezuela Tel 58.22.24.342 x248 Fax 58.22.24.7389 West Africa Sobidis Tel 225.20.33.40.32 Fax 225.20.33.40.28 04-7900030-2A