Download BD FastImmune™ CD63/CD123/Anti-HLA-DR

Monoclonal
Antibodies
Detecting
Human
Antigens
RESEARCH
APPLICATIONS
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BD FastImmune™
CD63/CD123/Anti–HLA-DR
Catalog No. 341068
50 Tests
20 µL/test
Research applications include studies of:
•
Basophils in whole blood1-4
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Basophil degranulation when activated5,6,7
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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, LAMP3, 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 granulocytemacrophage 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
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11/2014
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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
Immunofluorescence
activation
1. Add 20 µL of basophil stimulation buffer (BSB)5 with or without allergens onto the
bottom of the 12 x 75-mm tube.
•
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
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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).
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Figure 1 Gating strategy for basophil identification
104
0
100
R1
100
R1 gated
104
CD123 PE
R2
CD123 PE
SSC-H
1000
ungated
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).
2.18%
0.36%
CD63 FITC
104
sensitive
100
104
CD63 FITC
not sensitive
71.24%
CD63 FITC
104
100
100
CD123 PE
CD123 PE
53.09%
100
104
negative control (buffer)
104
CD123 PE
100
100
100
100
positive stimulus control (fMLP)
104
CD63 FITC
not sensitive
91.51%
104
104
sensitive
0.70%
100
CD63 FITC
104
100
CD123 PE
CD123 PE
100
not sensitive
104
sensitive
CD123 PE
104
Figure 2 Typical FL1 histograms and FL1 vs FL2 dot plots
grass pollen mix
100
CD63 FITC
104
HANDLING AND
STORAGE
Store vials at 2°C–8°C. Conjugated forms should not be frozen. Protect from exposure
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.
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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.
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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.
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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.
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Olweus J, BitMansour A, Warnke R, et al. Dendritic cell ontogeny: a human dendritic cell lineage of
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Azorsa DO, Hyman JA, Hildreth JEK. CD63/Pltgp40: a platelet activation antigen identical to the stagespecific, 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
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1995:11.
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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.
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18. Hart DNJ, McKenzie JL. Isolation and characterization of human tonsil dendritic cells. J Exp Med.
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cells develop into dendritic cells with interleukin (IL)-3 and CD40-ligand. J Exp Med. 1997;185:11011111.
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
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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 biotinavidin-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
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30. Protection of Laboratory Workers from Occupationally Acquired Infections; Approved Guideline —
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PATENTS AND
TRADEMARKS
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