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    Purified Mouse Anti-Human TNF for ICC
    Purified Mouse Anti-Human TNF for ICC
    PBMC were isolated from human peripheral blood by density gradient centrifugation and were cultured for 2 days with plate bound anti-human CD3 and soluble anti-human CD28 in the presence of human recombinant IL-2 and human recombinant IL-4. The cells were subsequently harvested, washed and recultured with human recombinant IL-2 and human recombinant IL-4 for an additional 3 days. Finally, the cells were harvested, washed and stimulated with PMA (Sigma, Cat. No. P-8139, 5 ng/ml) and ionomycin (Sigma, Cat. No. I-0634, 500 ng/ml) in the presence of GolgiStop™ (Cat. No. 554724) for 4 hr at 37°C. The activated cells were harvested and the presence of TNF producing cells was detected by immunocytochemistry using a three-step staining procedure that employs a Biotin Goat anti-Mouse IgG secondary antibody (Cat. No. 550337) and a horseradish peroxidase-based detection system (Streptavidin-HRP Cat. No. 550946) (Nomarski optics, original magnification 400X). To demonstrate specificity of staining, the binding of the Purified Mouse Anti-Human TNF for ICC (Cat. No. 559071) was blocked by the preincubation of the purified antibody with excess recombinant human TNF protein (Cat. No. 554589; data not shown).
    Purified Mouse Anti-Human TNF for ICC
    PBMC were isolated from human peripheral blood by density gradient centrifugation and were cultured for 2 days with plate bound anti-human CD3 and soluble anti-human CD28 in the presence of human recombinant IL-2 and human recombinant IL-4. The cells were subsequently harvested, washed and recultured with human recombinant IL-2 and human recombinant IL-4 for an additional 3 days. Finally, the cells were harvested, washed and stimulated with PMA (Sigma, Cat. No. P-8139, 5 ng/ml) and ionomycin (Sigma, Cat. No. I-0634, 500 ng/ml) in the presence of GolgiStop™ (Cat. No. 554724) for 4 hr at 37°C. The activated cells were harvested and the presence of TNF producing cells was detected by immunocytochemistry using a three-step staining procedure that employs a Biotin Goat anti-Mouse IgG secondary antibody (Cat. No. 550337) and a horseradish peroxidase-based detection system (Streptavidin-HRP Cat. No. 550946) (Nomarski optics, original magnification 400X). To demonstrate specificity of staining, the binding of the Purified Mouse Anti-Human TNF for ICC (Cat. No. 559071) was blocked by the preincubation of the purified antibody with excess recombinant human TNF protein (Cat. No. 554589; data not shown).
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    BD Pharmingen™
    Tumor necrosis factor alpha; TNF-a; TNF-α; TNFSF2; Cachectin
    Human (QC Testing), Rhesus,Cynomolgus,Baboon (Tested in Development)
    Mouse IgG1, κ
    Recombinant Human TNF
    Intracellular block/flow cytometry (Routinely Tested)
    0.5 mg/ml
    AB_397191
    Aqueous buffered solution containing ≤0.09% sodium azide.
    RUO


    Preparation And Storage

    Store undiluted at 4°C. The monoclonal antibody was purified from tissue culture supernatant or ascites by affinity chromatography.
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    Recommended Assay Procedures

    Immunocytochemistry:Purified Mouse Anti-Human TNF for ICC (Cat. No. 559071) antibody can be used to identify and enumerate human TNF producing cells by immunocytochemistry. For optimal indirect immunocytochemical staining, the antibody should be titrated and visualized via a three-step staining procedure. Please see protocol below for a detailed description of our recommended procedure.

    CYTOKINE IMMUNOCYTOCHEMISTRY PROTOCOL

    REAGENTS REQUIRED

    1. Fixation Buffer: 5% formalin (10% formalin, CMS, Cat. No. 245-684) is dissolved in phosphate buffered-saline (PBS) (Bacto  FA Buffer, Difco Laboratories, Cat. No.  2314-15-0), or BD Pharmingen™ ICC Fixation Buffer (BD Cat. No. 550010)

    2. Endogenous Peroxidase Blocking Buffer: DAKO Peroxidase Blocking Reagent (DAKO, Cat. No. S2001).

    3. Endogenous Biotin Blocking Buffer: Biotin/Avidin Blocking Kit (Vector Laboratories, Cat. No. SP-2001).

    4. Antibody dilution buffer: BD Pharmingen™ IHC Antibody Diluent Buffer supplemented with saponin (Cat. No. 559148).

    5. Microscopic slides: Adhesion Slides (Erie Scientific Company, Cat. No. ER-202B-AD) or for cytospins, Colorfrost /Plus slides (Fisher, Cat. No. 12-550-17).

    6. Detection system: BD Pharmingen™ Streptavidin-horseradish peroxidase (HRP), (Cat. No. 550946), or Anti-Mouse Ig HRP Detection Kit (Cat. No. 551011).  

    7. Mounting medium for short-term storage: Aqua-mount®  (Lerner Laboratories, Cat. No. 13800).

    8. DAB Substrate Kit (contains 3-3 -Diaminobenzidine tetra hydrochloride), (BD Cat. No. 550880), or Anti-Mouse Ig HRP Detection Kit (Cat. No. 551011).

     

    SECONDARY ANTIBODIES

    Biotin Goat anti-Mouse IgG (Cat. No. 550337) or Anti-Mouse Ig HRP Detection Kit (Cat. No. 551011)

    PROCEDURE FOR IMMUNOCYTOCHEMICAL STAINING OF SINGLE-CELL PREPARATIONS

    This procedure describes the immunoenzymatic technique of staining cytokines within individual cells that are immobilized on microscopic slides via adherence (adherent slides) or centrifugation (cytospins).  

    ADHESION SLIDES

    1. Harvest cells and wash them twice in PBS using centrifugation (400 x g for 5 min) to remove residual protein.

    2. Adjust the cell concentration to 4-5 × 10×6 cells/ml in PBS.

    3. Place 20 µl of the cell suspension in each well of the adhesion slides and let them adhere at room temperature (RT) for 20 min. Please note that the slides should be washed in PBS at RT for 5 min before transferring the cells.

    4. Fix cells on slides using fixation buffer for 15 min at RT.

    5. Wash slides 2X in PBS with 5 min incubations.

    6. Block slides with PBS supplemented with 1% (w/v) BSA (Sigma, Cat. No. A43-78) for 30 min at RT or 10 min at 37°C.

    7. Wash slides 2X in PBS and proceed with staining or air dry them and store them at -80°C for future use.

    8. Incubate slides with 20 µl of 1% goat serum and PBS with 0.1% (w/v) saponin for 30 min at RT.

    9. Wash slides 2X with PBS with 5 min incubations.

    10. Block endogenous peroxidase activity with Endogenous Peroxidase Blocking Buffer (20 µl/well) for 10 min at RT.

    11. Wash 2X in PBS with 5 min incubations.

    12. Incubate each well with Avidin (20 µl/well) for 15 min.

    13. Wash 2X in PBS with 5 min incubations.

    14. Incubate each well with Biotin (20 µl/well) for 15 min.

    15. Wash 2X in PBS with 5 min incubations.

    16. Incubate each well for 1 hr at RT with 20 µl of purified cytokine-specific antibody or appropriate immunoglobulin isotype control diluted in IHC Diluent Buffer (Cat. No. 559148) supplemented with saponin.

    17. Wash slides 2X in PBS with 5 min incubations.

    18. Incubate each well with 20 µl of a biotinylated secondary antibody diluted in IHC Diluent Buffer for 30 min at RT.

    19. Wash 2X in PBS with 5 min incubations.

    20. Apply 20 µl of Streptavidin-HRP (BD Cat. No. 550946)  to each well on slides and incubate for 30 min at RT.

    21. Wash slides 2X with PBS with 5 minutes incubations.

    22. Incubate with DAB Substrate as directed, (BD Cat. No. 550880) for less than 5 min at RT.

    23. Stop the development of the color reaction by washing with PBS.

    24. The slides are subsequently mounted in short-term storage mounting medium.

     

    CYTOSPINS

    1. Assemble the Cytospin's sample chamber (e.g. Cytospin 3, Shandon, UK or comparable centrifuge), filter card, slide and cytospin racks according to manufacturer's specifications.

    2. Load 40 µl of approximately 1 x 10×6 cells to each sample chamber.

    3. Spin slides at 600 rpm for 2 min.

    4. Take slides out of the cytospin rack and place them on a staining rack.

    5. For fixation and staining please follow the steps 4 through 24 specified above for staining cells on adhesion slides.

     

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    Product Notices

    1. Since applications vary, each investigator should titrate the reagent to obtain optimal results.
    2. 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.
    3. Sodium azide is a reversible inhibitor of oxidative metabolism; therefore, antibody preparations containing this preservative agent must not be used in cell cultures nor injected into animals. Sodium azide may be removed by washing stained cells or plate-bound antibody or dialyzing soluble antibody in sodium azide-free buffer. Since endotoxin may also affect the results of functional studies, we recommend the NA/LE (No Azide/Low Endotoxin) antibody format, if available, for in vitro and in vivo use.
    4. Species cross-reactivity detected in product development may not have been confirmed on every format and/or application.
    5. Please refer to http://regdocs.bd.com to access safety data sheets (SDS).
    6. Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
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    559071 Rev. 5
    Antibody Details
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    MAb11

    The MAb11 monoclonal antibody specifically binds to human tumor necrosis factor (TNF, also known as TNF-α) protein. TNF is an efficient juxtacrine, paracrine and endocrine mediator of inflammatory and immune functions. It regulates the growth and differentiation of a variety of cell types. TNF is cytotoxic for transformed cells when in conjunction with IFN-γ. It is secreted by activated monocytes/macrophages and other cells such as B cells, T cells and fibroblasts. The immunogen used to generate the MAb11 hybridoma was recombinant human TNF. The MAb11 antibody has been reported to crossreact with Rhesus Macaque TNF.

    559071 Rev. 5
    Format Details
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    Purified
    Tissue culture supernatant is purified by either protein A/G or affinity purification methods. Both methods yield antibody in solution that is free of most other soluble proteins, lipids, etc. This format provides pure antibody that is suitable for a number of downstream applications including: secondary labeling for flow cytometry or microscopy, ELISA, Western blot, etc.
    Purified
    559071 Rev.5
    Citations & References
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    Development References (8)

    1. Danis VA, Franic GM, Rathjen DA, Brooks PM. Effects of granulocyte-macrophage colony-stimulating factor (GM-CSF), IL-2, interferon-gamma (IFN-gamma), tumour necrosis factor-alpha (TNF-alpha) and IL-6 on the production of immunoreactive IL-1 and TNF-alpha by human monocytes. Clin Exp Immunol. 1991; 85(1):143-150. (Clone-specific: ELISA). View Reference
    2. Hsu SM, Raine L, Fanger H. A comparative study of the peroxidase-antiperoxidase method and an avidin-biotin complex method for studying polypeptide hormones with radioimmunoassay antibodies. Am J Clin Pathol. 1981; 75(5):734-738. (Methodology: Immunocytochemistry (cytospins)). View Reference
    3. Hsu SM, Raine L, Fanger H. Use of avidin-biotin-peroxidase complex (ABC) in immunoperoxidase techniques: a comparison between ABC and unlabeled antibody (PAP) procedures. J Histochem Cytochem. 1981; 29(4):577-580. (Methodology: Immunocytochemistry (cytospins)). View Reference
    4. Jason J, Larned J. Single-cell cytokine profiles in normal humans: comparison of flow cytometric reagents and stimulation protocols. J Immunol Methods. 1997; 207(1):13-22. (Biology). View Reference
    5. Rathjen DA, Cowan K, Furphy LJ, Aston R. Antigenic structure of human tumour necrosis factor: recognition of distinct regions of TNF alpha by different tumour cell receptors. Mol Immunol. 1991; 28(1-2):79-86. (Clone-specific: ELISA). View Reference
    6. Sander B, Andersson J, Andersson U. Assessment of cytokines by immunofluorescence and the paraformaldehyde-saponin procedure. Immunol Rev. 1991; 119:65-93. (Biology). View Reference
    7. Sopper S, Stahl-Hennig C, Demuth M, Johnston IC, Dorries R, ter Meulen V. Lymphocyte subsets and expression of differentiation markers in blood and lymphoid organs of rhesus monkeys. Cytometry. 1997; 29(4):351-362. (Biology). View Reference
    8. Verdier F, Aujoulat M, Condevaux F, Descotes J. Determination of lymphocyte subsets and cytokine levels in cynomolgus monkeys. Toxicology. 1995; 105(1):81-90. (Biology). View Reference
    View All (8) View Less
    559071 Rev. 5

    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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