Flow cytometric analysis of CD154 expression on stimulated human peripheral blood lymphocytes. Human peripheral blood mononuclear cells were stimulated (4 hours) with Phorbol 12-Myristate 13-Acetate (PMA; Sigma, Cat. No. P-8139; 20 ng/ml final concentration) and Ionomycin (Sigma, Cat. No. I-0634; 250 ng/ml final concentration). The cells were then stained with either APC-Cy7 Mouse IgG1, κ Isotype Control (Cat. No. 557873; dashed line histogram) or APC-Cy7 Mouse Anti-Human CD154 antibody (Cat. No. 563588; solid line histogram). The fluorescence histograms were derived from gated events with the forward and side light-scatter characteristics of viable activated lymphocytes. Flow cytometric analysis was performed using a BD™ LSR II Flow Cytometer System.
Flow cytometric analysis of CD154 expression on stimulated human peripheral blood lymphocytes. Human peripheral blood mononuclear cells were stimulated (4 hours) with Phorbol 12-Myristate 13-Acetate (PMA; Sigma, Cat. No. P-8139; 20 ng/ml final concentration) and Ionomycin (Sigma, Cat. No. I-0634; 250 ng/ml final concentration). The cells were then stained with either APC-Cy7 Mouse IgG1, κ Isotype Control (Cat. No. 557873; dashed line histogram) or APC-Cy7 Mouse Anti-Human CD154 antibody (Cat. No. 563588; solid line histogram). The fluorescence histograms were derived from gated events with the forward and side light-scatter characteristics of viable activated lymphocytes. Flow cytometric analysis was performed using a BD™ LSR II Flow Cytometer System.
Product Details
BD Pharmingen™
CD40LG; CD40 ligand; CD40L; TNFSF5; T-BAM; TRAP
Human (QC Testing), Rhesus, Cynomolgus, Baboon (Tested in Development)
Mouse BALB/c IgG1, κ
Human TRAP1 Transfected Cell Line
Flow cytometry (Routinely Tested)
5 µl
VI 6T-068
959
AB_2738296
Aqueous buffered solution containing BSA, protein stabilizer, 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 APC-Cy7 under optimum conditions, and unconjugated antibody and free APC-Cy7 were removed.
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).
- Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
- APC-Cy7 is a tandem fluorochrome composed of Allophycocyanin (APC), which is excited by laser lines between 595 and 647 nm and serves as an energy donor, coupled to the cyanine dye Cy7™, which acts as an energy acceptor and fluoresces at 780 nm. BD Biosciences Pharmingen has maximized the fluorochrome energy transfer in APC-Cy7, thus maximizing its fluorescence emission intensity, minimizing residual emission from APC, and minimizing required electronic compensation in multilaser-laser flow cytometry systems. Note: Although every effort is made to minimize the lot-to-lot variation in residual emission from APC, it is strongly recommended that every lot be tested for differences in the amount of compensation required and that individual compensation controls are run for each APC-Cy7 conjugate.
- APC-Cy7 tandem fluorochrome emission is collected in a detector for fluorescence wavelengths of 750 nm and higher.
- Warning: Some APC-Cy7 and PE-Cy7 conjugates show changes in their emission spectrum with prolonged exposure to formaldehyde. If you are unable to analyze fixed samples within four hours, we recommend that you use BD™ Stabilizing Fixative (Cat. No. 338036).
- 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.
- For fluorochrome spectra and suitable instrument settings, please refer to our Multicolor Flow Cytometry web page at www.bdbiosciences.com/colors.
- 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.
- Species cross-reactivity detected in product development may not have been confirmed on every format and/or application.
- An isotype control should be used at the same concentration as the antibody of interest.
Companion Products
Stain Buffer (FBS) RUO
Size
500 mL
Cat No.
554656
APC-Cy™7 Mouse IgG1, κ Isotype Control RUO
Size
100 Tests
Cat No.
557873
563588 Rev. 2
Antibody Details
TRAP1
The TRAP1 monoclonal antibody specifically binds to CD154. CD154 is a 39 kDa type II membrane glycoprotein that is a member of the tumor necrosis factor superfamily, Tumor necrosis factor ligand superfamily member 5 (TNFSF5). CD154 is expressed on a variety of cell types including activated CD4+ T cells and some CD8+ T cells, NK cells, mast cells and basophils. CD154 is also known as CD40 ligand (CD40L); it serves as a ligand for CD40 that is expressed on B cells, macrophages, and dendritic cells. The expression of CD154 by activated T-helper cells costimulates B-cell activation and proliferation through binding to CD40 expressed on B cells. In response to T-dependent antigens, the CD154 and CD40 interaction is required for B-lymphocyte differentiation, including immunoglobulin production and isotype switching and memory B cell generation. The TRAP1 antibody can partially block T cell-B cell interaction and inhibit the subsequent proliferation, differentiation, and memory formation of B cells. It has been reported that patients with X-linked hyper-IgM syndrome have defective expression of functional CD154 due to mutations in the CD40LG gene that encodes CD154.
563588 Rev. 2
Format Details
APC-Cy7
APC-Cy7 dye is a part of the BD APC red family of dyes. This tandem fluorochrome is comprised of a Allophycocyanin (APC) donor that has excitation maxima (Ex Max) of 651 nm and an acceptor dye, Cy™7, with an emission maximum (Em Max) at 779 nm. APC-Cy7 can be excited by the Red (627-640 nm) laser and detected using an optical filter centered near 780 nm (e.g., a 760/60 nm bandpass filter). Please ensure that your instrument’s configurations (lasers and optical filters) are appropriate for this dye.
APC-Cy7
Red 627-640 nm
651 nm
779 nm
563588 Rev.2
Citations & References
View product citations for antibody "563588" on CiteAb
Development References (12)
-
Altenburg A, Baldus SE, Smola H, Pfister H, Hess S. CD40 ligand-CD40 interaction induces chemokines in cervical carcinoma cells in synergism with IFN-gamma. J Immunol. 1999; 162(7):4140-4147. (Clone-specific: Immunohistochemistry). View Reference
-
Fuleihan R, Ramesh N, Horner A, et al. Cyclosporin A inhibits CD40 ligand expression in T lymphocytes. J Clin Invest. 1994; 93(3):1315-1320. (Biology). View Reference
-
Graf D, Muller S, Korthauer U, van Kooten C, Weise C, Kroczek RA. A soluble form of TRAP (CD40 ligand) is rapidly released after T cell activation. Eur J Immunol. 1995; 25(6):1749-1754. (Immunogen: Flow cytometry, Immunoprecipitation). View Reference
-
Gray D, Dullforce P, Jainandunsing S. Memory B cell development but not germinal center formation is impaired by in vivo blockade of CD40-CD40 ligand interaction. J Exp Med. 1994; 180(1):141-155. (Biology). View Reference
-
Henn V, Slupsky JR, Grafe M, et al. CD40 ligand on activated platelets triggers an inflammatory reaction of endothelial cells. Nature. 1998; 391(6667):591-594. (Clone-specific: Fluorescence microscopy, Immunofluorescence, Immunohistochemistry). View Reference
-
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.
-
Kroczek RA, Graf D, Brugnoni D, et al. Defective expression of CD40 ligand on T cells causes "X-linked immunodeficiency with hyper-IgM (HIGM1)". Immunol Rev. 1994; 138:39-59. (Clone-specific: Flow cytometry). View Reference
-
MacDonald KP, Nishioka Y, Lipsky PE, Thomas R. Functional CD40 ligand is expressed by T cells in rheumatoid arthritis. J Clin Invest. 1997; 100(9):2404-2414. (Clone-specific: Flow cytometry). View Reference
-
Mason D. David Mason .. et al., ed. Leucocyte typing VII : white cell differentiation antigens : proceedings of the Seventh International Workshop and Conference held in Harrogate, United Kingdom. Oxford: Oxford University Press; 2002.
-
Nishioka Y, Lipsky PE. The role of CD40-CD40 ligand interaction in human T cell-B cell collaboration. J Immunol. 1994; 153(3):1027-1036. (Biology). View Reference
-
O'Gorman MR, Zaas D, Paniagua M, Corrochano V, Scholl PR, Pachman LM. Development of a rapid whole blood flow cytometry procedure for the diagnosis of X-linked hyper-IgM syndrome patients and carriers. Clin Immunol Immunopathol. 1997; 85(2):172-181. (Biology: Flow cytometry). View Reference
-
van Kooten C, Gaillard C, Galizzi JP, et al. B cells regulate expression of CD40 ligand on activated T cells by lowering the mRNA level and through the release of soluble CD40. Eur J Immunol. 1994; 24(4):787-792. (Biology). View Reference
563588 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.