Two-color flow cytometric analysis of IL-12 (p40/p70) expression in stimulated human monocytes. Human peripheral blood mononuclear cells (PBMC) were preincubated (2 hr) with Recombinant Human IFN-γ (Cat. No. 554617/554616; 10 ng/ml). Lipopolysaccharide (LPS; Sigma, Cat. No. L-2654; 1.0 μg/ml) and BD GolgiStop™ Protein Transport Inhibitor (containing Monensin) (Cat. No. 554724) were then added to the IFN-γ-primed cells which were further cultured overnight. The cells were harvested, washed with BD Pharmingen™ Stain Buffer (FBS) (Cat. No. 554656), stained with APC Mouse Anti-Human CD14 antibody (Cat. No. 555399/561383/561708), and fixed with BD Cytofix™ Fixation Buffer (Cat. No. 554655). The cells were then permeabilized and stained in BD Perm/Wash™ Buffer (Cat. No. 554723) with either BD Horizon™ BV421 Mouse IgG1 κ Isotype Control (Cat. No. 562438; Left Panel) or BD Horizon BV421 Mouse Anti-Human IL-12 (p40/p70) antibody (Cat. No. 565023; Right Panel) using BD Biosciences Intracellular Cytokine Staining protocol. Two-color flow cytometric contour plots showing the correlated expression patterns of IL-12 (p40/p70) (or Ig Isotype control staining) versus CD14 were derived from gated events with the forward and side light-scatter characteristics of intact monocytes. Flow cytometric analysis was performed using a BD LSRFortessa™ Cell Analyzer System.
Two-color flow cytometric analysis of IL-12 (p40/p70) expression in stimulated human monocytes. Human peripheral blood mononuclear cells (PBMC) were preincubated (2 hr) with Recombinant Human IFN-γ (Cat. No. 554617/554616; 10 ng/ml). Lipopolysaccharide (LPS; Sigma, Cat. No. L-2654; 1.0 μg/ml) and BD GolgiStop™ Protein Transport Inhibitor (containing Monensin) (Cat. No. 554724) were then added to the IFN-γ-primed cells which were further cultured overnight. The cells were harvested, washed with BD Pharmingen™ Stain Buffer (FBS) (Cat. No. 554656), stained with APC Mouse Anti-Human CD14 antibody (Cat. No. 555399/561383/561708), and fixed with BD Cytofix™ Fixation Buffer (Cat. No. 554655). The cells were then permeabilized and stained in BD Perm/Wash™ Buffer (Cat. No. 554723) with either BD Horizon™ BV421 Mouse IgG1 κ Isotype Control (Cat. No. 562438; Left Panel) or BD Horizon BV421 Mouse Anti-Human IL-12 (p40/p70) antibody (Cat. No. 565023; Right Panel) using BD Biosciences Intracellular Cytokine Staining protocol. Two-color flow cytometric contour plots showing the correlated expression patterns of IL-12 (p40/p70) (or Ig Isotype control staining) versus CD14 were derived from gated events with the forward and side light-scatter characteristics of intact monocytes. Flow cytometric analysis was performed using a BD LSRFortessa™ Cell Analyzer System.
Product Details
BD Horizon™
IL12B; IL-12B; IL12p40; IL-12-40; CLMF; CLMF2; NKSF; NKSF2
Human (QC Testing)
Mouse IgG1, κ
Purified p40 Subunit of Human IL-12
Intracellular staining (flow cytometry) (Routinely Tested)
5 µl
AB_2739045
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 BD Horizon BV421 under optimum conditions, and unconjugated antibody and free BD Horizon BV421 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).
- An isotype control should be used at the same concentration as the antibody of interest.
- 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.
- Source of all serum proteins is from USDA inspected abattoirs located in the United States.
- Pacific Blue™ is a trademark of Molecular Probes, Inc., Eugene, OR.
- For fluorochrome spectra and suitable instrument settings, please refer to our Multicolor Flow Cytometry web page at www.bdbiosciences.com/colors.
- Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
Companion Products
Brilliant Stain Buffer RUO
Size
100 Tests
Cat No.
563794
Stain Buffer (FBS) RUO
Size
500 mL
Cat No.
554656
Stain Buffer (BSA) RUO
Size
500 mL
Cat No.
554657
Fixation Buffer RUO
Size
100 mL
Cat No.
554655
Perm/Wash Buffer RUO
Size
100 mL
Cat No.
554723
Protein Transport Inhibitor (Containing Monensin) RUO
Size
0.7 mL
Cat No.
554724
565023 Rev. 1
Antibody Details
C8.6
The C8.6 antibody reacts with human IL-12 p40 monomers, homodimers, and p70 heterodimers, but not p35 subunits. The immunogen used to generate the C8.6 hybridoma was the purified p40 subunit of human IL-12. The C8.6 antibody is a neutralizing antibody and has been reported to strongly inhibit three different biological activities of IL-12 (i.e., IFN-γ induction), mitogenic effects on PHA blasts, and enhancement of NK cell-mediated cytotoxicity. C8.6 has been shown to immunoprecipitate IL-23.
The antibody was conjugated to BD Horizon BV421 which is part of the BD Horizon Brilliant™ Violet family of dyes. With an Ex Max of 407-nm and Em Max at 421-nm, BD Horizon BV421 can be excited by the violet laser and detected in the standard Pacific Blue™ filter set (eg, 450/50-nm filter). BD Horizon BV421 conjugates are very bright, often exhibiting a 10 fold improvement in brightness compared to Pacific Blue conjugates.
565023 Rev. 1
Format Details
BV421
The BD Horizon Brilliant Violet™ 421 (BV421) Dye is part of the BD Horizon Brilliant Violet™ family of dyes. This polymer-technology based dye has an excitation maximum (Ex Max) of 407-nm and an emission maximum (Em Max) at 423-nm. Driven by BD innovation, BV421 is designed to be excited by the violet laser (405-nm) and detected using an optical filter centered near 420-nm (e.g., a 431/28-nm or 450/50-nm bandpass filter). BV421 is an ideal alternative for V450 as it is approximately ten times brighter with less spillover into the BV510/V500 detector. Please ensure that your instrument’s configurations (lasers and optical filters) are appropriate for this dye.
BV421
Violet 405 nm
407 nm
423 nm
565023 Rev.1
Citations & References
View product citations for antibody "565023" on CiteAb
Development References (3)
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D'Andrea A, Aste-Amezaga M, Valiante NM, Ma X, Kubin M, Trinchieri G. Interleukin 10 (IL-10) inhibits human lymphocyte interferon gamma-production by suppressing natural killer cell stimulatory factor/IL-12 synthesis in accessory cells. J Exp Med. 1993; 178(3):1041-1048. (Clone-specific: Neutralization). View Reference
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D'Andrea A, Rengaraju M, Valiante NM, et al. Production of natural killer cell stimulatory factor (interleukin 12) by peripheral blood mononuclear cells. J Exp Med. 1992; 176(5):1387-1398. (Clone-specific). View Reference
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Oppmann B, Lesley R, Blom B, et al. Novel p19 protein engages IL-12p40 to form a cytokine, IL-23, with biological activities similar as well as distinct from IL-12.. Immunity. 2000; 13(5):715-25. (Clone-specific: Immunoprecipitation). View Reference
565023 Rev. 1
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.