Flow cytometric analysis of CD63 expression on human peripheral blood platelets. Platelets were isolated from fresh whole blood and activated by Thrombin (Sigma-Aldrich, Cat. No.T8885), and then fixed with 2% formaldehyde. After washing, the fixed platelets were stained with either PerCP-Cy™5.5 Mouse IgG1 κ Isotype Control (Cat. No. 550795; dashed line histogram) or PerCP-Cy™5.5 Mouse Anti-Human CD63 antibody (Cat. No. 565426; solid line histogram). The fluorescence histogram showing CD63 expression (or Ig Isotype control staining) was derived from gated events with the forward and side light-scatter characteristics of intact platelets. Flow cytometry was performed using a BD™ LSR II Flow Cytometer System.
Flow cytometric analysis of CD63 expression on human peripheral blood platelets. Platelets were isolated from fresh whole blood and activated by Thrombin (Sigma-Aldrich, Cat. No.T8885), and then fixed with 2% formaldehyde. After washing, the fixed platelets were stained with either PerCP-Cy™5.5 Mouse IgG1 κ Isotype Control (Cat. No. 550795; dashed line histogram) or PerCP-Cy™5.5 Mouse Anti-Human CD63 antibody (Cat. No. 565426; solid line histogram). The fluorescence histogram showing CD63 expression (or Ig Isotype control staining) was derived from gated events with the forward and side light-scatter characteristics of intact platelets. Flow cytometry was performed using a BD™ LSR II Flow Cytometer System.
Product Details
BD Pharmingen™
LAMP-3; ME491; MLA-1; Granulophysin; Ptgr40; NGA; gp55
Human (QC Testing), Rhesus, Cynomolgus, Baboon (Tested in Development)
Mouse BALB/c IgG1, κ
Human Splenic Adherent Cells
Flow cytometry (Routinely Tested)
5 µl
V P036
967
AB_2739233
Aqueous buffered solution containing BSA and ≤0.09% sodium azide.
RUO
Preparation And Storage
Store undiluted at 4°C and protected from prolonged exposure to light. Do not freeze. The monoclonal antibody was purified from tissue culture supernatant or ascites by affinity chromatography. The antibody was conjugated with PerCP-Cy5.5 under optimum conditions, and unconjugated antibody and free PerCP-Cy5.5 were removed. Storage of PerCP-Cy5.5 conjugates in unoptimized diluent is not recommended and may result in loss of signal intensity.
Product Notices
- This reagent has been pre-diluted for use at the recommended Volume per Test. We typically use 1 × 10^6 cells in a 100-µl experimental sample (a test).
- An isotype control should be used at the same concentration as the antibody of interest.
- Source of all serum proteins is from USDA inspected abattoirs located in the United States.
- Caution: Sodium azide yields highly toxic hydrazoic acid under acidic conditions. Dilute azide compounds in running water before discarding to avoid accumulation of potentially explosive deposits in plumbing.
- Please observe the following precautions: Absorption of visible light can significantly alter the energy transfer occurring in any tandem fluorochrome conjugate; therefore, we recommend that special precautions be taken (such as wrapping vials, tubes, or racks in aluminum foil) to prevent exposure of conjugated reagents, including cells stained with those reagents, to room illumination.
- PerCP-Cy5.5–labelled antibodies can be used with FITC- and R-PE–labelled reagents in single-laser flow cytometers with no significant spectral overlap of PerCP-Cy5.5, FITC, and R-PE fluorescence.
- PerCP-Cy5.5 is optimized for use with a single argon ion laser emitting 488-nm light. Because of the broad absorption spectrum of the tandem fluorochrome, extra care must be taken when using dual-laser cytometers, which may directly excite both PerCP and Cy5.5™. We recommend the use of cross-beam compensation during data acquisition or software compensation during data analysis.
- Species cross-reactivity detected in product development may not have been confirmed on every format and/or application.
- For fluorochrome spectra and suitable instrument settings, please refer to our Multicolor Flow Cytometry web page at www.bdbiosciences.com/colors.
- Cy is a trademark of GE Healthcare.
- Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
Companion Products
Stain Buffer (FBS) RUO
Size
500 mL
Cat No.
554656
Stain Buffer (BSA) RUO
Size
500 mL
Cat No.
554657
PerCP-Cy™5.5 Mouse IgG1 κ Isotype Control RUO
Size
0.1 mg
Cat No.
550795
565426 Rev. 2
Antibody Details
H5C6
The H5C6 monoclonal antibody specifically binds to CD63. CD63 is a 53 kDa, type III lysosomal glycoprotein, expressed on activated platelets, monocytes and macrophages. This molecule is also referred to in the literature as LIMP, gp55, melanoma-associated antigen ME491, Pltgp40, LAMP-3 and is a member of the tetraspan transmembrane 4 superfamily (TM4SF). It is widely expressed on surface and in the cytoplasm of various hematopoietic (monocytes, macrophages) and non-hematopoietic (endothelium, fibroblasts, osteoclasts, smooth muscle) cells. CD63 plays roles in mediating cellular adhesion and motility.
565426 Rev. 2
Format Details
PerCP-Cy5.5
PerCP-Cy5.5 dye is part of the BD blue family of dyes. This tandem fluorochrome is comprised of a fluorescent protein complex (PerCP) with an excitation maximum (Ex Max) of 482 nm and an acceptor dye with an emission maximum (Em Max) at 676 nm. PerCP-Cy5 is designed to be excited by the blue laser (488-nm) and detected using an optical filter centered near 680 nm (e.g., a 695/40 nm bandpass filter). The donor dye can be partially excited by the Violet (405-nm) laser resulting in cross-laser excitation and fluorescence spillover. Please ensure that your instrument’s configurations (lasers and optical filters) are appropriate for this dye.
PerCP-Cy5.5
Blue 488 nm
482 nm
676 nm
565426 Rev.2
Citations & References
Development References (4)
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Azorsa DO, Hyman JA, Hildreth JE. CD63/Pltgp40: a platelet activation antigen identical to the stage-specific, melanoma-associated antigen ME491. Blood. 1991; 78(2):280-284. (Clone-specific: Immunoprecipitation). View Reference
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Hildreth JE, Derr D, Azorsa DO. Characterization of a novel self-associating Mr 40,000 platelet glycoprotein. Blood. 1991; 77(1):121-132. (Immunogen: Flow cytometry, Immunohistochemistry, Immunoprecipitation, Radioimmunoassay). View Reference
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Kishimoto T. Tadamitsu Kishimoto .. et al., ed. Leucocyte typing VI : white cell differentiation antigens : proceedings of the sixth international workshop and conference held in Kobe, Japan, 10-14 November 1996. New York: Garland Pub.; 1997.
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Schlossman SF. Stuart F. Schlossman .. et al., ed. Leucocyte typing V : white cell differentiation antigens : proceedings of the fifth international workshop and conference held in Boston, USA, 3-7 November, 1993. Oxford: Oxford University Press; 1995.
565426 Rev. 2
Please refer to Support Documents for Quality Certificates
Global - Refer to manufacturer's instructions for use and related User Manuals and Technical data sheets before using this products as described
Comparisons, where applicable, are made against older BD Technology, manual methods or are general performance claims. Comparisons are not made against non-BD technologies, unless otherwise noted.
For Research Use Only. Not for use in diagnostic or therapeutic procedures.
