Description
The B27 monoclonal antibody specifically binds to human interferon-γ (IFN-γ), a 14-18 kDa glycoprotein containing 143 amino acid residues. IFN-γ is a potent multifunctional cytokine produced by several activated cell types including NK, NKT, CD4+TCRαβ+, CD8+TCRαβ+, and TCRγδ+ T cells. IFN-γ exerts its biological effects through specific binding to the high-affinity IFN-γ receptor complex comprised of IFN-γRα (CD119) and IFN-γRβ subunits. In addition to its antiviral effects, IFN-γ upregulates a number of lymphoid cell functions including the antimicrobial and anti-tumor responses of macrophages, NK cells, and neutrophils. In addition, IFN-γ influences the regulation of proliferation, differentiation, and effector responses of B cell and T cell subsets. These influences can involve IFN-γ's capacity to boost MHC class I and II expression by antigen-presenting cells as well as direct effects on B cells and T cells themselves. B27 is a neutralizing antibody. The use of B27 antibody for epitope mapping of human IFN-γ has been described. The B27 antibody has been reported not to bind to denatured IFN-γ.
Immunofluorescent Staining and Flow Cytometric Analysis: The FITC-, PE-, APC-, PE-Cy7-, Alexa Fluor® 647-, and Alexa Fluor® 488-conjugated B27 antibody (Cat. No. 554700, 554701, 554702, 557643, 557729, and 557718) is useful for multicolor immunofluorescent staining and flow cytometric analysis to identify and enumerate IFN-γ producing cells within mixed cell populations. For optimal immunofluorescent staining this antibody (Cat. No. 557718) should be used at 5 µl per test. A useful control for demonostrating specificity of staining is the following: pre-block the paraformaldehyde-fixed/saponin-permeabilized cells with unlabeled B27 antibody (Cat. No. 554699) prior to staining. The intracellular cytokine staining technique and blocking controls are described in detail by C. Prussin and D. Metcalfe. A suitable mouse IgG1 isotype control for assessing the level of background staining on paraformaldehyde-fixed/saponin-permeabilized human cells is Alexa Fluor® 488-MOPC-21 (Cat. No. 557721); use at 5 µl/test. Donor-to-donor variations will occur and should be taken into account when analyzing cytokine expression. For specific methodology and additional information on intracellular staining, please refer to the protocols under "Intracellular Flow" at our website at http://www.bdbiosciences.com/us/s/resources. Neutralization: The NA/LE™ B27 antibody (Cat. No. 554698) is useful for neutralization of human IFN-γ bioactivity. A suitable NA/LE™ mouse IgG1 isotype control to match the NA/LE B27 antibody is the 107.3 antibody, Cat. No. 554721. IP/WB: The PE-conjugated B27 antibody has been reported to be useful for immunoprecipitation studies. The B27 antibody has been reported not to bind to denatured IFN-γ. Please note that this antibody is not routinely tested at BD Biosciences Pharmingen.
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 to Alexa Fluor® 488 under optimum conditions, and unreacted Alexa Fluor® 488 was removed.
1. 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).
2. Source of all serum proteins is from USDA inspected abattoirs located in the United States.
3. 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.
4. For fluorochrome spectra and suitable instrument settings, please refer to our Multicolor Flow Cytometry web page at www.bdbiosciences.com/colors.
5. Alexa Fluor® 488 fluorochrome emission is collected at the same instrument settings as for fluorescein isothiocyanate (FITC).
6. The Alexa Fluor®, Pacific Blue™, and Cascade Blue® dye antibody conjugates in this product are sold under license from Molecular Probes, Inc. for research use only, excluding use in combination with microarrays, or as analyte specific reagents. The Alexa Fluor® dyes (except for Alexa Fluor® 430), Pacific Blue™ dye, and Cascade Blue® dye are covered by pending and issued patents.
7. Alexa Fluor® is a registered trademark of Molecular Probes, Inc., Eugene, OR.
8. Species cross-reactivity detected in product development may not have been confirmed on every format and/or application.
9. Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
