Flow cytometric analysis of intracellular IL-12 p40/p70 expression by activated mouse macrophages. Thioglycollate-elicited mouse peritoneal macrophages were primed with recombinant mouse IFN-γ (10 ng/ml, Cat. No. 554587) for 2 hr and stimulated overnight with lipopolysaccharide (LPS, Sigma, Cat. No. L-8272; 1 μg/ml) and BD GolgiPlug™ Protein Transport Inhibitor (Containing Brefeldin A) (Cat. No. 555029). The adherent cells were washed with 1× phosphate buffered saline (PBS) and incubated with 1× trypsin-EDTA solution (37°C, 15 min). The cells were harvested, washed, incubated with Fc Block™ (Rat IgG2b,κ Anti-Mouse CD16/CD32) antibody (Cat. No. 553142), fixed and permeabilized using BD Cytofix™ Fixation Buffer (Cat. No. 554655) and BD Perm/Wash™ Buffer (Cat. No. 554723). The cells were then stained either with a BD Horizon™ V450 Rat IgG1, κ Isotype Control (Cat No. 560535, Left Panel) or with the BD Horizon™ V450 Rat Anti-Mouse IL-12 (p40/p70) antibody (Cat No. 561456, Right Panel). MiCK-3 Mouse Cytokine Positive Control Cells (Cat No. 554654) are prepared in a similar manner. These cells can be used as a positive control for cytokine flow cytometry experiments designed to characterize the nature of mouse IL-12-producing cells. Two-color flow cytometric dot plots showing the correlated expression of IL-12 (or Ig Isotype control staining) versus cellular autofluorescence measured in the phycoerythrin channel (autofluorescence) were derived from events with the forward and side light-scatter characteristics of intact macrophages. Flow cytometry was performed using a BD™ LSR II Flow Cytometer System.
Flow cytometric analysis of intracellular IL-12 p40/p70 expression by activated mouse macrophages. Thioglycollate-elicited mouse peritoneal macrophages were primed with recombinant mouse IFN-γ (10 ng/ml, Cat. No. 554587) for 2 hr and stimulated overnight with lipopolysaccharide (LPS, Sigma, Cat. No. L-8272; 1 μg/ml) and BD GolgiPlug™ Protein Transport Inhibitor (Containing Brefeldin A) (Cat. No. 555029). The adherent cells were washed with 1× phosphate buffered saline (PBS) and incubated with 1× trypsin-EDTA solution (37°C, 15 min). The cells were harvested, washed, incubated with Fc Block™ (Rat IgG2b,κ Anti-Mouse CD16/CD32) antibody (Cat. No. 553142), fixed and permeabilized using BD Cytofix™ Fixation Buffer (Cat. No. 554655) and BD Perm/Wash™ Buffer (Cat. No. 554723). The cells were then stained either with a BD Horizon™ V450 Rat IgG1, κ Isotype Control (Cat No. 560535, Left Panel) or with the BD Horizon™ V450 Rat Anti-Mouse IL-12 (p40/p70) antibody (Cat No. 561456, Right Panel). MiCK-3 Mouse Cytokine Positive Control Cells (Cat No. 554654) are prepared in a similar manner. These cells can be used as a positive control for cytokine flow cytometry experiments designed to characterize the nature of mouse IL-12-producing cells. Two-color flow cytometric dot plots showing the correlated expression of IL-12 (or Ig Isotype control staining) versus cellular autofluorescence measured in the phycoerythrin channel (autofluorescence) were derived from events with the forward and side light-scatter characteristics of intact macrophages. Flow cytometry was performed using a BD™ LSR II Flow Cytometer System.
Product Details
BD Horizon™
IL-12/IL-23 p40; Il12b; Interleukin 12b; CLMF p40
Mouse (QC Testing)
Rat IgG1
CHO-expressed recombinant mouse IL-12 p70 protein
Intracellular staining (flow cytometry) (Routinely Tested)
0.2 mg/ml
AB_10714785
Aqueous buffered solution containing protein stabilizer 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 BD Horizon™ V450 under optimum conditions, and unreacted BD Horizon™ V450 was removed. Store undiluted at 4°C and protected from prolonged exposure to light. Do not freeze.
Product Notices
- Since applications vary, each investigator should titrate the reagent to obtain optimal results.
- An isotype control should be used at the same concentration as the antibody of interest.
- BD Horizon V450 has a maximum absorption of 406 nm and maximum emission of 450 nm. Before staining with this reagent, please confirm that your flow cytometer is capable of exciting the fluorochrome and discriminating the resulting fluorescence.
- Pacific Blue™ is a trademark of Molecular Probes, Inc., Eugene, OR.
- 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.
- 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
Purified Rat Anti-Mouse CD16/CD32 (Mouse BD Fc Block™) RUO
Size
0.5 mg
Cat No.
553142
Stain Buffer (FBS) RUO
Size
500 mL
Cat No.
554656
Fixation Buffer RUO
Size
100 mL
Cat No.
554655
Perm/Wash Buffer RUO
Size
100 mL
Cat No.
554723
V450 Rat IgG1, κ Isotype Control RUO
Size
0.1 mg
Cat No.
560535
MiCK-3 Mouse Cytokine Positive Control Cells RUO
Size
1 mL
Cat No.
554654
561456 Rev. 1
Antibody Details
C15.6
The C15.6 monoclonal antibody specifically binds to both free and complexed (homodimer p80 and heterodimer p70) forms of the p40 subunit of mouse interleukin-12 (IL-12). The immunogen used to generate the C15.6 hybridoma was recombinant mouse IL-12 p70 protein. p40 has also been described as a subunit of IL-23 and thus it is possible that the C15.6 antibody will crossreact with IL-23.
The antibody is conjugated to BD Horizon™ V450, which has been developed for use in multicolor flow cytometry experiments and is available exclusively from BD Biosciences. It is excited by the Violet laser Ex max of 406 nm and has an Em Max at 450 nm. Conjugates with BD Horizon™ V450 can be used in place of Pacific Blue™ conjugates.
561456 Rev. 1
Format Details
V450
BD Horizon™ V450 Dye is part of the BD Horizon™ violet family of dyes. This is a small organic fluorochrome with an excitation maximum (Ex Max) at 405-nm and an emission maximum (Em Max) at 450-nm. BD Horizon™ V450, driven by BD innovation, is designed to be excited by the violet laser (405 nm) and detected using an optical filter centered near 450-nm (e.g., a 450/50-nm bandpass filter). The dye can be excited by the UV (355-nm) laser resulting in cross-laser excitation and spillover. Please ensure that your instrument’s configurations (lasers and optical filters) are appropriate for this dye.
V450
Violet 405 nm
405 nm
450 nm
561456 Rev.1
Citations & References
Development References (8)
-
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. (Biology). View Reference
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Gately MK, Chizzonite R, Presky DH. Measurement of Human and Mouse Interleukin-12. In: Cooligan J, Kruisbeek A, Margulies D, Shevach E, Storber W, ed. Current Protocols in Immunology. New York: John Wiley and Sons; 1995:6-16.
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Gately MK, Renzetti LM, Magram J, et al. The interleukin-12/interleukin-12-receptor system: role in normal and pathologic immune responses. Annu Rev Immunol. 1998; 16:495-521. (Biology). View Reference
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Maruo S, Ahn HJ, Yu WG, et al. Establishment of an IL-12-responsive T cell clone: its characterization and utilization in the quantitation of IL-12 activity. J Leukoc Biol. 1997; 61(3):346-352. (Biology). View Reference
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Neurath MF, Fuss I, Kelsall BL, Stuber E, Strober W. Antibodies to interleukin 12 abrogate established experimental colitis in mice. J Exp Med. 1995; 182(5):1281-1290. (Biology). View Reference
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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: Flow cytometry). View Reference
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Wu C, Ferrante J, Gately MK, Magram J. Characterization of IL-12 receptor beta1 chain (IL-12Rbeta1)-deficient mice: IL-12Rbeta1 is an essential component of the functional mouse IL-12 receptor. J Immunol. 1997; 159(4):1658-1665. (Biology). View Reference
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Wysocka M, Kubin M, Vieira LQ, et al. Interleukin-12 is required for interferon-gamma production and lethality in lipopolysaccharide-induced shock in mice. Eur J Immunol. 1995; 25(3):672-676. (Biology). View Reference
561456 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.
