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      FITC Mouse Anti-Rat IFN-γ
      FITC Mouse Anti-Rat IFN-γ
      Expression of IFN-γ by stimulated LOU rat  lymphoid cells. Lymphoid cells from LOU rat were stimulated for 2 days with plate bound purified NA/LE™ mouse anti-rat CD3 (10  µg/ml; Cat. No. 554829), purified NA/LE™ mouse anti-rat CD28 (2 µg/ml; Cat. No.554993), recombinant rat IL-2 (10 ng/ml; Cat. No.555106) and recombinant rat IL-4 (50 ng/ml; Cat. No. 555107). The cells were then incubated for 3 days with recombinant rat IL-2 and IL-4. Following the 3 day incubation, the lymphoid cells were restimulated for 4 hours with PMA (5 ng/ml final concentration, Sigma, Cat. No.P-8139) and ionomycin (500 ng/ml final concentration, Sigma, Cat. No.I-0634) in the presence of GolgiPlug™ (1 µl/ml, Cat. No.555029). The activated cells were harvested, fixed, permeabilized, and subsequently stained with 20 µl of FITC- conjugated mouse anti-rat IFN- antibody (FITC-DB-1, Cat. No. 559498) by using Pharmingen's staining protocol (see Center panel). To demonstrate specificity of  staining, the binding by the FITC-DB-1 antibody was blocked by pre-incubation of the fixed/permeabilized cells with unlabeled DB-1 antibody (5 µg; Right panel) prior to staining. The quadrant markers for the bivariate dot plots were set based on the staining profile using FITC-MOPC-21 isotype control antibody (Cat. No. 554679, see Left panel) and  verified using the unlabeled antibody blocking specificity control.
      FITC Mouse Anti-Rat IFN-γ
      Expression of IFN-γ by stimulated LOU rat  lymphoid cells. Lymphoid cells from LOU rat were stimulated for 2 days with plate bound purified NA/LE™ mouse anti-rat CD3 (10  µg/ml; Cat. No. 554829), purified NA/LE™ mouse anti-rat CD28 (2 µg/ml; Cat. No.554993), recombinant rat IL-2 (10 ng/ml; Cat. No.555106) and recombinant rat IL-4 (50 ng/ml; Cat. No. 555107). The cells were then incubated for 3 days with recombinant rat IL-2 and IL-4. Following the 3 day incubation, the lymphoid cells were restimulated for 4 hours with PMA (5 ng/ml final concentration, Sigma, Cat. No.P-8139) and ionomycin (500 ng/ml final concentration, Sigma, Cat. No.I-0634) in the presence of GolgiPlug™ (1 µl/ml, Cat. No.555029). The activated cells were harvested, fixed, permeabilized, and subsequently stained with 20 µl of FITC- conjugated mouse anti-rat IFN- antibody (FITC-DB-1, Cat. No. 559498) by using Pharmingen's staining protocol (see Center panel). To demonstrate specificity of  staining, the binding by the FITC-DB-1 antibody was blocked by pre-incubation of the fixed/permeabilized cells with unlabeled DB-1 antibody (5 µg; Right panel) prior to staining. The quadrant markers for the bivariate dot plots were set based on the staining profile using FITC-MOPC-21 isotype control antibody (Cat. No. 554679, see Left panel) and  verified using the unlabeled antibody blocking specificity control.
      Product Details
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      BD Pharmingen™
      Rat (QC Testing)
      Mouse IgG1, κ
      Recombinant Rat IFN-γ
      Intracellular staining (flow cytometry) (Routinely Tested)
      20 µl
      25712
      AB_397253
      Aqueous buffered solution containing BSA and ≤0.09% sodium azide.
      RUO


      Preparation And Storage

      The monoclonal antibody was purified from tissue culture supernatant or ascites by affinity chromatography. The antibody was conjugated with FITC under optimum conditions, and unreacted FITC was removed. Store undiluted at 4°C and protected from prolonged exposure to light. Do not freeze.
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      Recommended Assay Procedures

      Immunofluorescent Staining and Flow Cytometric Analysis: The DB-1 antibody is useful for immunofluorescent staining and flow cytometric analysis to identify and enumerate IFN-γ producing cells within mixed cell populations. This 100 Test Size formulation of the FITC-conjugated DB-1 antibody has been pre-titrated to assure effective intracellular detection of rat IFN-γ using 20 µl/1 x 10^6 cells in a final volume of 100 µl. For specific methodology, please visit our web site, www.bdbiosciences.com, and go to the protocols section or the chapter on intracellular staining in the Immune Function Handbook. A useful control for demonstrating specificity of staining is to pre-block the fixed/permeabilized cells with unlabeled DB-1 antibody prior to staining. The intracellular staining technique and the use of blocking controls have been 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/saponinpermeabilized rat cells is also available: FITC-MOPC-21 (Cat. No. 554679).

      Important Note: This pre-titered antibody solution does not contain a cell permeabilization agent. It is necessary to include a cell permeabilization agent when using the pre-titered antibody solution to stain fixed and permeabilized cells. Perm/Wash™ Buffer (Cat. No. 554723) contains the permeabiliization agent saponin and is useful for this purpose as described below.

      Resuspend one million fixed and permeabilized cells in 20 µl of the pre-titered antibody solution and 30 µl of 1X Perm/Wash™ Buffer (Cat. No. 554723). Incubate the cell suspension for 15 minutes (at RT or 4°C). Wash twice in 100 µl of 1X Perm/Wash™ Buffer (Cat. No. 554723).

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      Product Notices

      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. Since applications vary, each investigator should titrate the reagent to obtain optimal results.
      3. Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
      4. Source of all serum proteins is from USDA inspected abattoirs located in the United States.
      5. 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.
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      559498 Rev. 2
      Antibody Details
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      DB-1

      The DB-1 monoclonal antibody specifically binds to rat interferon-γ (IFN-γ).  The immunogen used to generate the DB-1 hybridoma was recombinant rat IFN-γ expressed in COS cells.  This is a neutralizing antibody.  

      559498 Rev. 2
      Format Details
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      FITC
      Fluorescein (FITC) is part of the BD blue family of dyes. This is a small organic fluorochrome with an excitation maximum (Ex Max) at 494-nm and an emission maximum (Em Max) at 518-nm. FITC is designed to be excited by the Blue laser (488-nm) and detected using an optical filter centered near 520 nm (e.g., a 530/30-nm bandpass filter). Please ensure that your instrument’s configurations (lasers and optical filters) are appropriate for this dye.
      altImg
      FITC
      Blue 488 nm
      494 nm
      518 nm
      559498 Rev.2
      Citations & References
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      Development References (7)

      1. Bakhiet M, Olsson T, Mhlanga J. Human and rodent interferon-gamma as a growth factor for Trypanosoma brucei. Eur J Immunol. 1996; 26(6):1359-1364. (Biology). View Reference
      2. Prussin C, Metcalfe DD. Detection of intracytoplasmic cytokine using flow cytometry and directly conjugated anti-cytokine antibodies. J Immunol Methods. 1995; 188(1):117-128. (Methodology). View Reference
      3. Schmidt B, Stoll G, van der Meide P, Jung S, Hartung HP. Transient cellular expression of gamma-interferon in myelin-induced and T-cell line-mediated experimental autoimmune neuritis. 1992; 115(Pt 6):1633-1646. (Biology). View Reference
      4. van der Meide PH, Borman AH, Beljaars HG, Dubbeld MA, Botman CA, Schellekens H. Isolation and characterization of monoclonal antibodies directed to rat interferon-gamma. Leuk Res. 1989; 8(4):439-449. (Biology). View Reference
      5. van der Meide PH, Borman TH, de Labie MC, et al. A sensitive two-site enzyme immunoassay for the detection of rat interferon-gamma in biological fluids. J Interferon Res. 1990; 10(2):183-189. (Biology). View Reference
      6. van der Meide PH, Dubbeld M, Vijverberg K, Kos T, Schellekens H. The purification and characterization of rat gamma interferon by use of two monoclonal antibodies.. J Gen Virol. 1986; 67(Pt 6):1059-1071. (Biology). View Reference
      7. van der Meide PH, Groenestein RJ, de Labie MC, Aten J, Weening JJ. Susceptibility to mercuric chloride-induced glomerulonephritis is age-dependent: study of the role of IFN-gamma. Cell Immunol. 1995; 162(1):131-137. (Biology). View Reference
      View All (7) View Less
      559498 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.

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