BD Fastimmune™ CD63/CD123/Anti-HLA-DR

• • Monoclonal • BD FastImmune™ Antibodies • Detecting • CD63/CD123/Anti–HLA-DR Human • • Catalog No. 341068 50 Tests 20 µL/test Antigens • • • • •

RESEARCH Research applications include studies of: APPLICATIONS • Basophils in whole blood1-4 • Basophil degranulation when activated5,6,7 • Dendritic cell subsets in peripheral blood and lymphoid tissues8

DESCRIPTION Specificity The CD63 antibody reacts with a 53-kilodalton (kDa), type III lysosomal glycoprotein found in many cell types, including monocytes, platelets, and basophils. This molecule is also referred to as LIMP, gp55, melanoma-associated antigen ME491, Pltgp40, LAMP- 3, and is a member of the tetraspannin transmembrane 4 superfamily (TM4SF).9-12 The CD123 antibody (Anti–IL-3Rα) binds to the α-chain of the interleukin-3 receptor (IL-3Rα).13 It selectively recognizes COS cells that have been transfected with IL-3Rα. Clone 9F5 does not block IL-3 binding to the receptor.14 Anti–HLA-DR recognizes a human class II major histocompatibility complex (MHC) antigen.15,16 The antigen is a transmembrane glycoprotein composed of α and β subunits that have molecular weights of 36 and 27 kilodaltons (kDa), respectively.15,16 Antigen distribution The CD63 antibody is an intracellular, lysosomal protein whose surface expression is upregulated on activated platelets, degranulated neutrophils, monocytes, macrophages, endothelium, and activated basophils.9-12,5,6,17 The CD123 antibody is expressed on a subset of peripheral blood dendritic cells,8,18-20 on a subset of progenitor cells,8 monocytes,14 eosinophils,14 and basophils.1-4 Neutrophils show IL-3Rα expression when they are cultured with granulocyte- macrophage colony-stimulating factor (GM-CSF) but not when freshly isolated.21 Since IL-3 stimulates the production of hematopoietic cells, such as megakaryocytes, erythroid cells, and B cells, it is assumed that precursor cells of these lineages can also express IL-3Rα.14 HLA-DR is expressed on B lymphocytes, monocytes, macrophages, activated T lymphocytes, activated natural killer (NK) lymphocytes, and human progenitor cells.22-26 It is also present on thymic epithelium, B-lymphocyte–dependent areas of spleen and lymph node, and B-cell lymphomas.26-29 Clones The CD63 antibody, clone H5C6, is derived from the fusion of P3x653 Ag8 myeloma cells with splenocytes from BALB/c mice immunized with human splenic adherent cells.9,10 The CD123 antibody, clone 9F5,13 is derived from fusion of NS-1 cells with splenocytes from BALB/c mice immunized with IL-3Rα transfected COS cells.14

For Research Use Only. Not for use in diagnostic or therapeutic procedures.

Becton, Dickinson and Company BD Biosciences 2350 Qume Drive San Jose, CA 95131 USA

bdbiosciences.com [email protected] 11/2014 23-6584-02 Anti–HLA-DR, clone L243, is derived from the hybridization of NS-1/1-Ag4 mouse myeloma cells with spleen cells from BALB/c mice immunized with the human lymphoblastic B-cell line RPMI 8866.15

Composition The CD63 and CD123 antibodies are each composed of mouse IgG1 heavy chains and kappa light chains.

Anti–HLA-DR is composed of mouse IgG2a heavy chains and kappa light chains. The BD FastImmune™ CD63/CD123/Anti–HLA-DR reagent is supplied as a combination of CD63 FITC, CD123 PE, and Anti–HLA-DR PerCP in 1.0 mL of phosphate-buffered saline (PBS) containing bovine serum albumin (BSA) and 0.1% sodium azide.

PROCEDURE Visit our website (bdbiosciences.com) or contact your local BD representative for the lyse/wash protocol for direct immunofluorescence. Method for direct 1. Add 20 µL of basophil stimulation buffer (BSB)5 with or without allergens onto the Immunofluorescence bottom of the 12 x 75-mm tube. activation • negative stimulus control — working BSB only (no allergens) • positive stimulus control — diluted fMLP (N-Formyl-Met-Leu-Phe) in BSB at 2-µM concentration • test sample — diluted grass pollen mix/mite mix, or other allergen in BSB 2. Aliquot 100 µL of blood (with heparin anticoagulant) to all stimulation tubes, vortex, and incubate the samples in a 37°C water bath for 10–15 minutes. 3. Stop the degranulation immediately by transferring sample tubes to an ice bath or by adding 10 µL of 20-mM EDTA at room temperature for 5 minutes. Staining 1. Add 20 µL of CD63 FITC/CD123 PE/Anti–HLA-DR PerCP antibody cocktail to each tube, vortex, and incubate in the dark on ice for 20 minutes or at room temperature for 15 minutes (when using EDTA to stop the reaction). 2. Lyse samples with 2 mL of 1X BD FACS™ lysing solution at room temperature for 15 minutes. 3. Centrifuge samples at 300 x g for 5 minutes, and aspirate the supernatant. 4. Wash again with 1–2 mL of PBS. 5. Add 0.3 mL of 0.5% paraformaldehyde (PFA) to resuspend samples. NOTE Samples are ready for flow cytometric analysis.

REPRESENTATIVE DATA Flow cytometric analysis was performed on lysed whole blood. Samples were analyzed on a BD FACS™ flow cytometer with a 488-nm laser. Data was acquired with a threshold on FL2 set to eliminate most of CD123-negative cells, and at least 500 CD123-positive cells per sample were acquired. Basophils were identified as low side scatter (SSC), CD123-positive and HLA-DR–negative7,8 cells (Figure 1). The quantitative determination of activated or degranulated basophils was measured on CD63 FITC (FL1). Markers were set by the negative control samples and can be shown as an FL1 vs FL2 dot plot (see Figure 2).

23-6584-02 Page 2 Figure 1 Gating strategy for basophil identification

R1 gated ungated 4 10

1000 R2

R1 SSC-H CD123 PE 0 0 4 10 100 CD123 PE 10 100 Anti–HLA-DR PerCP 104 Figure 2 displays representative data from a donor sensitive to grass pollens and a donor not sensitive to grass pollens. Samples with buffer, with a positive stimulus control (fMLP), or with grass pollen mix were incubated at 37°C for 10 minutes. Basophils were gated with R1 and R2 (see Figure 1).

Figure 2 Typical FL1 histograms and FL1 vs FL2 dot plots

sensitive not sensitive 4 4 10 10 2.18% 0.36%

negative control (buffer) CD123 PE CD123 PE CD123 0 0 10 10 100 CD63 FITC 104 100 CD63 FITC 104

sensitive not sensitive 4 4 10 10 53.09% 71.24%

positive stimulus control (fMLP) CD123 PE CD123 CD123 PE CD123 0 0 10 10 100 CD63 FITC 104 100 CD63 FITC 104 sensitive not sensitive 4 4 10 91.51% 10 0.70%

grass pollen mix CD123 PE CD123 PE CD123 0 0 10 10 100 CD63 FITC 104 100 CD63 FITC 104

HANDLING AND Store vials at 2°C–8°C. Conjugated forms should not be frozen. Protect from exposure STORAGE to light. Each reagent is stable until the expiration date shown on the bottle label when stored as directed.

WARNING All biological specimens and materials coming in contact with them are considered biohazards. Handle as if capable of transmitting infection30,31 and dispose of with proper precautions in accordance with federal, state, and local regulations. Never pipette by mouth. Wear suitable protective clothing, eyewear, and gloves.

Page 3 23-6584-02 CHARACTERIZATION To ensure consistently high-quality reagents, each lot of antibody is tested for conformance with characteristics of a standard reagent. Representative flow cytometric data is included in this data sheet.

WARRANTY Unless otherwise indicated in any applicable BD general conditions of sale for non-US customers, the following warranty applies to the purchase of these products.

THE PRODUCTS SOLD HEREUNDER ARE WARRANTED ONLY TO CONFORM TO THE QUANTITY AND CONTENTS STATED ON THE LABEL OR IN THE PRODUCT LABELING AT THE TIME OF DELIVERY TO THE CUSTOMER. BD DISCLAIMS HEREBY ALL OTHER WARRANTIES, EXPRESSED OR IMPLIED, INCLUDING WARRANTIES OF MERCHANTABILITY AND FITNESS FOR ANY PARTICULAR PURPOSE AND NONINFRINGEMENT. BD’S SOLE LIABILITY IS LIMITED TO EITHER REPLACEMENT OF THE PRODUCTS OR REFUND OF THE PURCHASE PRICE. BD IS NOT LIABLE FOR PROPERTY DAMAGE OR ANY INCIDENTAL OR CONSEQUENTIAL DAMAGES, INCLUDING PERSONAL INJURY, OR ECONOMIC LOSS, CAUSED BY THE PRODUCT.

REFERENCES 1. Agis H, Füreder W, Bankl HC, et al. Comparative immunophenotypic analysis of human mast cells, blood basophils and monocytes. Immunology. 1996;87:535-543.

2. Valent P. Immunophenotypic characterization of human basophils and mast cells. Chem Immunol. 1995;61:34-48.

3. Valent P, Besemer J, Muhm M, Majdic O, Lechner K, Bettelheim P. Interleukin 3 activates human blood basophils via high-affinity binding sites. Proc Natl Acad Sci USA. 1989;86:5542-5546.

4. Agis H, Beil WJ, Bankl HC, et al. Mast cell-lineage versus basophil lineage involvement in myeloproliferative and myelodysplastic syndromes: diagnostic role of cell-immunophenotyping. Leukemia and Lymphoma. 1996;22:187-204. 5. Sainte-Laudy J, Vallon C, Guerin JC. Diagnosis of latex allergy: comparison of histamine release and flow cytometric analysis of basophil activation. Inflamm Res. 1996;45:S35-S36.

6. Gane P, Pecquet C, Crespeau H, Lambin P, Leynadier F, Rouger P. Flow cytometric monitoring of allergen induced basophil activation. Cytometry. 1995;19:361-365.

7. Stain C, Stockinger H, Scharf M, et al. Human blood basophils display a unique phenotype including activation linked membrane structures. Blood. 1987;70:1872-1879. 8. Olweus J, BitMansour A, Warnke R, et al. Dendritic cell ontogeny: a human dendritic cell lineage of myeloid origin. Proc Natl Acad Sci USA. 1997;94:12551-12556.

9. Azorsa DO, Hyman JA, Hildreth JEK. CD63/Pltgp40: a platelet activation antigen identical to the stage- specific, melanoma-associated antigen ME491. Blood. 1991;78:280-284.

10. Hildreth JEK, Derr D, Azorsa DO. Characterization of a novel self-associating Mr 40,000 platelet glycoprotein. Blood. 1991;77:121-132. 11. Azorsa DO, Hildreth JEK. Cluster report: CD63. In: Schlossman SF, Boumsell L, Gilks W, et al, eds. Leucocyte Typing V: White Cell Differentiation Antigens. New York, NY: Oxford University Press; 1995:11.

12. de Haas M, Azorsa DO. CD guide: CD63. In: Kishimoto T, Kikutani H, von dem Borne AEGK, eds. Leucocyte Typing VI: White Cell Differentiation Antigens. New York, NY: Garland Publishing, Inc; 1997:1160-1161.

13. Miyajima A. CDw123 (interleukin 3 receptor α chain) Workshop Panel report. In: Kishimoto T, Kikutani H, von dem Borne AEGK, et al, eds. Leucocyte Typing VI: White Cell Differentiation Antigens. New York, NY: Garland Publishing, Inc; 1997:854-855.

14. Sun Q, Woodcock JM, Rapoport A, et al. Monoclonal antibody 7G3 recognizes the N-terminal domain of the human Interleukin-3 (IL-3) receptor α-chain and functions as a specific IL-3 receptor antagonist. Blood. 1996;87:83-92.

15. Lampson LA, Levy R. Two populations of Ia-like molecules on a human B cell line. J Immunol. 1980;125:293-299.

16. Brodsky FM. A matrix approach to human class II histocompatibility antigens: reactions of four monoclonal antibodies with the products of nine haplotypes. Immunogenetics. 1984;19:179-194. 17. Falcone FH, Haas H, Gibbs BF. The human basophil: a new appreciation of its role in immune responses. Blood. 2000;96:4028-4038.

23-6584-02 Page 4 18. Hart DNJ, McKenzie JL. Isolation and characterization of human tonsil dendritic cells. J Exp Med. 1988;168:157-170.

19. Grouard G, Rissoan M-C, Filgueira L, Durand I, Banchereau J, Liu Y-J. The enigmatic plasmocytoid T cells develop into dendritic cells with interleukin (IL)-3 and CD40-ligand. J Exp Med. 1997;185:1101- 1111.

20. O'Doherty U, Peng M, Gezelter S, et al. Human blood contains two subsets of dendritic cells, one immunologically mature and the other immature. Immunology. 1994;82:487-493. 21. Smith WB, Guida L, Sun Q, et al. Neutrophils activated by granulocyte-macrophage colony-stimulating factor express receptors for Interleukin-3 which mediate class II expression. Blood. 1995;86:3938-3944.

22. Tomkinson B, Wagner D, Nelson D, Sullivan J. Activated lymphocytes during acute Epstein-Barr virus infection. J Immunol. 1987;139:3802-3807.

23. Levacher M, Tallet S, Dazza M, Dournon E, Rouveix B, Pocidalo J. T activation marker evaluation in ARC patients treated with AZT: comparison with CD4+ lymphocyte count in non-progressors and progressors towards AIDS. Clin Exp Immunol. 1990;81:177-182.

24. Stites DP, Casavant CH, McHugh TM, et al. Flow cytometric analysis of lymphocyte phenotypes in AIDS using monoclonal antibodies and simultaneous dual immunofluorescence. Clin Immunol Immunopathol. 1986;38:161-177.

25. Terstappen LWMM, Hollander Z, Meiners H, Loken MR. Quantitative comparison of myeloid antigens on five lineages of mature peripheral blood cells. J Leuk Biol. 1990;48:138-148.

26. Warnke RA, Levy R. Detection of T and B antigens with hybridoma monoclonal antibodies: a biotin- avidin-horseradish peroxidase method. J Histochem Cytochem. 1980;28:771-776.

27. Warnke R, Miller R, Grogan T, Pederson M, Dilley J, Levy R. Immunologic phenotype in 30 patients with diffuse large-cell lymphoma. N Eng J Med. 1980;303:293-300.

28. Engleman EG, Warnke R, Fox RI, Dilley J, Benike CJ, Levy R. Studies of a human T lymphocyte antigen recognized by a monoclonal antibody. Proc Natl Acad Sci USA. 1981;78:1791-1795.

29. Zipf RF, Fox R, Dilley J, Levy R. Definition of the high risk ALL patient by immunologic phenotyping with monoclonal antibodies. Cancer Res. 1981;41:4786.

30. Protection of Laboratory Workers from Occupationally Acquired Infections; Approved Guideline — Third Edition. Wayne, PA: Clinical and Laboratory Standards Institute; 2005. CLSI document M29-A3. 31. Centers for Disease Control. Perspectives in disease prevention and health promotion update: universal precautions for prevention of transmission of human immunodeficiency virus, hepatitis B virus, and other bloodborne pathogens in health-care settings. MMWR. 1988;37:377-388.

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