BD Pharmingen™ Alexa Fluor® 488 Rat Anti-Mouse IL-4
Clone 11B11 (RUO)
Expression of IL-4 by stimulated CD4+ and CD4- C57BL/6 spleen cells. Splenocytes from C57BL/6 mice were enriched for CD4+ cells by positive selection using anti-CD4 coated plates (GK1.5 at10 µg/ml, Cat. No.5537256) for 1 hr at 4°C. Cells were harvested and stimulated with plate-bound anti-mouse CD3 (145-2C11,10 µg/ml, Cat. No. 553057) and soluble anti-CD28 (37.51 at 2 µg/ml, Cat. No. 553294) antibody in the presence of recombinant mouse IL-2 (10 ng/ml, Cat. No. 550069) and IL-4 (50 ng/ml, Cat. No. 550067) for 2 days. The cells were subsequently washed and expanded in IL-2 and IL-4 for 3 days. Following expansion the cells were washed and stimulated for 4 hrs with PMA (5 ng/ml, Sigma, Cat. No. P-8139) and ionomycin (500 ng, Sigma, P-8139) in the presence of BD GolgiPlug™, Cat. No. 555029). Following incubation the cells were harvested and stained with PE-anti-mouse CD4 (Cat. No. 553048) and either rat anti-mouse IL-4 (Alexa Fluor 488-11B11, Cat. No. 557728) (left panel) or immunoglobulin isotype control (Alexa Fluor 488-R3-34, Cat. No. 557720) (right panel) by using the BD Pharmingen staining protocol. To demonstrate specificity of staining the binding of Alexa Fluor™ 488 was blocked by the preincubation of the conjugated antibody with molar excess of recombinant mouse IL-4 (0.25 µg, Cat. No. 550067, data not shown) and by preincubation of the fixed/permeabilized cells with an excess of unlabelled 11B11 antibody (5 µg, Cat. No. 554433, data not shown) prior to stainining. The quadrant markers for the bivariate dot plots were set based on the autofluorescence and isotype controls.
Expression of IL-4 by stimulated CD4+ and CD4- C57BL/6 spleen cells. Splenocytes from C57BL/6 mice were enriched for CD4+ cells by positive selection using anti-CD4 coated plates (GK1.5 at10 µg/ml, Cat. No.5537256) for 1 hr at 4°C. Cells were harvested and stimulated with plate-bound anti-mouse CD3 (145-2C11,10 µg/ml, Cat. No. 553057) and soluble anti-CD28 (37.51 at 2 µg/ml, Cat. No. 553294) antibody in the presence of recombinant mouse IL-2 (10 ng/ml, Cat. No. 550069) and IL-4 (50 ng/ml, Cat. No. 550067) for 2 days. The cells were subsequently washed and expanded in IL-2 and IL-4 for 3 days. Following expansion the cells were washed and stimulated for 4 hrs with PMA (5 ng/ml, Sigma, Cat. No. P-8139) and ionomycin (500 ng, Sigma, P-8139) in the presence of BD GolgiPlug™, Cat. No. 555029). Following incubation the cells were harvested and stained with PE-anti-mouse CD4 (Cat. No. 553048) and either rat anti-mouse IL-4 (Alexa Fluor 488-11B11, Cat. No. 557728) (left panel) or immunoglobulin isotype control (Alexa Fluor 488-R3-34, Cat. No. 557720) (right panel) by using the BD Pharmingen staining protocol. To demonstrate specificity of staining the binding of Alexa Fluor™ 488 was blocked by the preincubation of the conjugated antibody with molar excess of recombinant mouse IL-4 (0.25 µg, Cat. No. 550067, data not shown) and by preincubation of the fixed/permeabilized cells with an excess of unlabelled 11B11 antibody (5 µg, Cat. No. 554433, data not shown) prior to stainining. The quadrant markers for the bivariate dot plots were set based on the autofluorescence and isotype controls.
Expression of IL-4 by stimulated CD4+ and CD4- C57BL/6 spleen cells. Splenocytes from C57BL/6 mice were enriched for CD4+ cells by positive selection using anti-CD4 coated plates (GK1.5 at10 µg/ml, Cat. No.5537256) for 1 hr at 4°C. Cells were harvested and stimulated with plate-bound anti-mouse CD3 (145-2C11,10 µg/ml, Cat. No. 553057) and soluble anti-CD28 (37.51 at 2 µg/ml, Cat. No. 553294) antibody in the presence of recombinant mouse IL-2 (10 ng/ml, Cat. No. 550069) and IL-4 (50 ng/ml, Cat. No. 550067) for 2 days. The cells were subsequently washed and expanded in IL-2 and IL-4 for 3 days. Following expansion the cells were washed and stimulated for 4 hrs with PMA (5 ng/ml, Sigma, Cat. No. P-8139) and ionomycin (500 ng, Sigma, P-8139) in the presence of BD GolgiPlug™, Cat. No. 555029). Following incubation the cells were harvested and stained with PE-anti-mouse CD4 (Cat. No. 553048) and either rat anti-mouse IL-4 (Alexa Fluor 488-11B11, Cat. No. 557728) (left panel) or immunoglobulin isotype control (Alexa Fluor 488-R3-34, Cat. No. 557720) (right panel) by using the BD Pharmingen staining protocol. To demonstrate specificity of staining the binding of Alexa Fluor™ 488 was blocked by the preincubation of the conjugated antibody with molar excess of recombinant mouse IL-4 (0.25 µg, Cat. No. 550067, data not shown) and by preincubation of the fixed/permeabilized cells with an excess of unlabelled 11B11 antibody (5 µg, Cat. No. 554433, data not shown) prior to stainining. The quadrant markers for the bivariate dot plots were set based on the autofluorescence and isotype controls.
Expression of IL-4 by stimulated CD4+ and CD4- C57BL/6 spleen cells. Splenocytes from C57BL/6 mice were enriched for CD4+ cells by positive selection using anti-CD4 coated plates (GK1.5 at10 µg/ml, Cat. No.5537256) for 1 hr at 4°C. Cells were harvested and stimulated with plate-bound anti-mouse CD3 (145-2C11,10 µg/ml, Cat. No. 553057) and soluble anti-CD28 (37.51 at 2 µg/ml, Cat. No. 553294) antibody in the presence of recombinant mouse IL-2 (10 ng/ml, Cat. No. 550069) and IL-4 (50 ng/ml, Cat. No. 550067) for 2 days. The cells were subsequently washed and expanded in IL-2 and IL-4 for 3 days. Following expansion the cells were washed and stimulated for 4 hrs with PMA (5 ng/ml, Sigma, Cat. No. P-8139) and ionomycin (500 ng, Sigma, P-8139) in the presence of BD GolgiPlug™, Cat. No. 555029). Following incubation the cells were harvested and stained with PE-anti-mouse CD4 (Cat. No. 553048) and either rat anti-mouse IL-4 (Alexa Fluor 488-11B11, Cat. No. 557728) (left panel) or immunoglobulin isotype control (Alexa Fluor 488-R3-34, Cat. No. 557720) (right panel) by using the BD Pharmingen staining protocol. To demonstrate specificity of staining the binding of Alexa Fluor™ 488 was blocked by the preincubation of the conjugated antibody with molar excess of recombinant mouse IL-4 (0.25 µg, Cat. No. 550067, data not shown) and by preincubation of the fixed/permeabilized cells with an excess of unlabelled 11B11 antibody (5 µg, Cat. No. 554433, data not shown) prior to stainining. The quadrant markers for the bivariate dot plots were set based on the autofluorescence and isotype controls.
Expression of IL-4 by stimulated CD4+ and CD4- C57BL/6 spleen cells. Splenocytes from C57BL/6 mice were enriched for CD4+ cells by positive selection using anti-CD4 coated plates (GK1.5 at10 µg/ml, Cat. No.5537256) for 1 hr at 4°C. Cells were harvested and stimulated with plate-bound anti-mouse CD3 (145-2C11,10 µg/ml, Cat. No. 553057) and soluble anti-CD28 (37.51 at 2 µg/ml, Cat. No. 553294) antibody in the presence of recombinant mouse IL-2 (10 ng/ml, Cat. No. 550069) and IL-4 (50 ng/ml, Cat. No. 550067) for 2 days. The cells were subsequently washed and expanded in IL-2 and IL-4 for 3 days. Following expansion the cells were washed and stimulated for 4 hrs with PMA (5 ng/ml, Sigma, Cat. No. P-8139) and ionomycin (500 ng, Sigma, P-8139) in the presence of BD GolgiPlug™, Cat. No. 555029). Following incubation the cells were harvested and stained with PE-anti-mouse CD4 (Cat. No. 553048) and either rat anti-mouse IL-4 (Alexa Fluor 488-11B11, Cat. No. 557728) (left panel) or immunoglobulin isotype control (Alexa Fluor 488-R3-34, Cat. No. 557720) (right panel) by using the BD Pharmingen staining protocol. To demonstrate specificity of staining the binding of Alexa Fluor™ 488 was blocked by the preincubation of the conjugated antibody with molar excess of recombinant mouse IL-4 (0.25 µg, Cat. No. 550067, data not shown) and by preincubation of the fixed/permeabilized cells with an excess of unlabelled 11B11 antibody (5 µg, Cat. No. 554433, data not shown) prior to stainining. The quadrant markers for the bivariate dot plots were set based on the autofluorescence and isotype controls.
Expression of IL-4 by stimulated CD4+ and CD4- C57BL/6 spleen cells. Splenocytes from C57BL/6 mice were enriched for CD4+ cells by positive selection using anti-CD4 coated plates (GK1.5 at10 µg/ml, Cat. No.5537256) for 1 hr at 4°C. Cells were harvested and stimulated with plate-bound anti-mouse CD3 (145-2C11,10 µg/ml, Cat. No. 553057) and soluble anti-CD28 (37.51 at 2 µg/ml, Cat. No. 553294) antibody in the presence of recombinant mouse IL-2 (10 ng/ml, Cat. No. 550069) and IL-4 (50 ng/ml, Cat. No. 550067) for 2 days. The cells were subsequently washed and expanded in IL-2 and IL-4 for 3 days. Following expansion the cells were washed and stimulated for 4 hrs with PMA (5 ng/ml, Sigma, Cat. No. P-8139) and ionomycin (500 ng, Sigma, P-8139) in the presence of BD GolgiPlug™, Cat. No. 555029). Following incubation the cells were harvested and stained with PE-anti-mouse CD4 (Cat. No. 553048) and either rat anti-mouse IL-4 (Alexa Fluor 488-11B11, Cat. No. 557728) (left panel) or immunoglobulin isotype control (Alexa Fluor 488-R3-34, Cat. No. 557720) (right panel) by using the BD Pharmingen staining protocol. To demonstrate specificity of staining the binding of Alexa Fluor™ 488 was blocked by the preincubation of the conjugated antibody with molar excess of recombinant mouse IL-4 (0.25 µg, Cat. No. 550067, data not shown) and by preincubation of the fixed/permeabilized cells with an excess of unlabelled 11B11 antibody (5 µg, Cat. No. 554433, data not shown) prior to stainining. The quadrant markers for the bivariate dot plots were set based on the autofluorescence and isotype controls.
Expression of IL-4 by stimulated CD4+ and CD4- C57BL/6 spleen cells. Splenocytes from C57BL/6 mice were enriched for CD4+ cells by positive selection using anti-CD4 coated plates (GK1.5 at10 µg/ml, Cat. No.5537256) for 1 hr at 4°C. Cells were harvested and stimulated with plate-bound anti-mouse CD3 (145-2C11,10 µg/ml, Cat. No. 553057) and soluble anti-CD28 (37.51 at 2 µg/ml, Cat. No. 553294) antibody in the presence of recombinant mouse IL-2 (10 ng/ml, Cat. No. 550069) and IL-4 (50 ng/ml, Cat. No. 550067) for 2 days. The cells were subsequently washed and expanded in IL-2 and IL-4 for 3 days. Following expansion the cells were washed and stimulated for 4 hrs with PMA (5 ng/ml, Sigma, Cat. No. P-8139) and ionomycin (500 ng, Sigma, P-8139) in the presence of BD GolgiPlug™, Cat. No. 555029). Following incubation the cells were harvested and stained with PE-anti-mouse CD4 (Cat. No. 553048) and either rat anti-mouse IL-4 (Alexa Fluor 488-11B11, Cat. No. 557728) (left panel) or immunoglobulin isotype control (Alexa Fluor 488-R3-34, Cat. No. 557720) (right panel) by using the BD Pharmingen staining protocol. To demonstrate specificity of staining the binding of Alexa Fluor™ 488 was blocked by the preincubation of the conjugated antibody with molar excess of recombinant mouse IL-4 (0.25 µg, Cat. No. 550067, data not shown) and by preincubation of the fixed/permeabilized cells with an excess of unlabelled 11B11 antibody (5 µg, Cat. No. 554433, data not shown) prior to stainining. The quadrant markers for the bivariate dot plots were set based on the autofluorescence and isotype controls.
Expression of IL-4 by stimulated CD4+ and CD4- C57BL/6 spleen cells. Splenocytes from C57BL/6 mice were enriched for CD4+ cells by positive selection using anti-CD4 coated plates (GK1.5 at10 µg/ml, Cat. No.5537256) for 1 hr at 4°C. Cells were harvested and stimulated with plate-bound anti-mouse CD3 (145-2C11,10 µg/ml, Cat. No. 553057) and soluble anti-CD28 (37.51 at 2 µg/ml, Cat. No. 553294) antibody in the presence of recombinant mouse IL-2 (10 ng/ml, Cat. No. 550069) and IL-4 (50 ng/ml, Cat. No. 550067) for 2 days. The cells were subsequently washed and expanded in IL-2 and IL-4 for 3 days. Following expansion the cells were washed and stimulated for 4 hrs with PMA (5 ng/ml, Sigma, Cat. No. P-8139) and ionomycin (500 ng, Sigma, P-8139) in the presence of BD GolgiPlug™, Cat. No. 555029). Following incubation the cells were harvested and stained with PE-anti-mouse CD4 (Cat. No. 553048) and either rat anti-mouse IL-4 (Alexa Fluor 488-11B11, Cat. No. 557728) (left panel) or immunoglobulin isotype control (Alexa Fluor 488-R3-34, Cat. No. 557720) (right panel) by using the BD Pharmingen staining protocol. To demonstrate specificity of staining the binding of Alexa Fluor™ 488 was blocked by the preincubation of the conjugated antibody with molar excess of recombinant mouse IL-4 (0.25 µg, Cat. No. 550067, data not shown) and by preincubation of the fixed/permeabilized cells with an excess of unlabelled 11B11 antibody (5 µg, Cat. No. 554433, data not shown) prior to stainining. The quadrant markers for the bivariate dot plots were set based on the autofluorescence and isotype controls.
Preparation And Storage
Recommended Assay Procedures
The PE-conjugated 11B11 antibody can be used for multicolor flow cytometric analyses to identify and enumerate IL-4 producing cells within mixed cell populations. For optimal immunofluorescent staining with flow cytometric analysis, this anti-cytokine antibody should be pretitrated (≤ 0.5 µg mAb/million cells). For specific methodology, please visit the protocols section or chapter on intracellular staining in the Immune Function Handbook, both of which are posted on our web site, www.bdbiosciences.com.
BD® CompBeads can be used as surrogates to assess fluorescence spillover (compensation). When fluorochrome conjugated antibodies are bound to BD® CompBeads, they have spectral properties very similar to cells. However, for some fluorochromes there can be small differences in spectral emissions compared to cells, resulting in spillover values that differ when compared to biological controls. It is strongly recommended that when using a reagent for the first time, users compare the spillover on cell and BD® CompBeads to ensure that BD® CompBeads are appropriate for your specific cellular application.
Product Notices
- Since applications vary, each investigator should titrate the reagent to obtain optimal results.
- Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
- Alexa Fluor® 488 fluorochrome emission is collected at the same instrument settings as for fluorescein isothiocyanate (FITC).
- For fluorochrome spectra and suitable instrument settings, please refer to our Multicolor Flow Cytometry web page at www.bdbiosciences.com/colors.
- 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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Companion Products
Interleukin-4 (IL-4) is a pleiotropic cytokine that has many roles, such as inducing the differentiation of naïve helper T cells (Th0 cells) to Th2 cells, stimulating activated B-cell and T-cell proliferation, and promoting immunoglobulin class switching to IgG1 and IgE in mouse B-cells. IL-4 is expressed by CD4 T-cells, mast cells, basophils and eosinophils. IL-4 was previously known as B-Cell Differentiation Factor (BCDF) or B-cell Stimulatory Factor (BSF1). The 11B11 monoclonal antibody specifically binds to mouse IL-4. The immunogen used to generate the 11B11 hybridoma was partially purified mouse IL-4 prepared from the supernatant of Phorbol 12-Myristate 13-Acetate (PMA)-stimulated EL-4 cells. The 11B11 antibody is reportedly a neutralizing antibody.
This antibody is routinely tested by flow cytometric analysis. Other applications were tested at BD Biosciences Pharmingen during antibody development only or reported in the literature.
Development References (4)
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Assenmacher M, Schmitz J, Radbruch A. Flow cytometric determination of cytokines in activated murine T helper lymphocytes: expression of interleukin-10 in interferon-gamma and in interleukin-4-expressing cells. Eur J Immunol. 1994; 24(5):1097-1101. (Clone-specific: Flow cytometry). View Reference
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Ohara J, Paul WE. Production of a monoclonal antibody to and molecular characterization of B-cell stimulatory factor-1. Nature. 1985; 315(6017):333-336. (Immunogen). View Reference
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Sander B, Andersson J, Andersson U. Assessment of cytokines by immunofluorescence and the paraformaldehyde-saponin procedure. Immunol Rev. 1991; 119:65-93. (Clone-specific: ELISA, Flow cytometry). View Reference
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Sander B, Hoiden I, Andersson U, Moller E, Abrams JS. Similar frequencies and kinetics of cytokine producing cells in murine peripheral blood and spleen. Cytokine detection by immunoassay and intracellular immunostaining. J Immunol Methods. 1993; 166(2):201-214. (Clone-specific: ELISA, Flow cytometry, Neutralization). View Reference
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