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Expression of IL-6 by stimulated BALB/c cells. BALB/c bone marrow cells were cultured for 2 weeks in the presence of recombinant mouse GM-CSF (20 ng/ml final concentration; Cat. No. 554586). The cells were washed and stimulated with recombinant mouse IFN-γ (10 ng/ml final concentration; Cat. No. 554587) for 4 hours followed by an overnight stimulation with LPS (1 µg/ml final concentration; Sigma, Cat. #L-8272) and GolgiPlug™ (1 µg/ml final concentration; Cat. No. 555029; aka Brefeldin A). Adherent cells were incubated with 1x trypsin EDTA at 37°C for 10 minutes and gently dislodged by pipetting for cell harvest. Nonspecific binding of cell surface antigens was blocked by incubation of the cells with Fc Block™ (10 µg/ml final concentration; Cat. No. 553142). The cells were fixed , permeabilized then subsequently stained with 0.06 µg of PE-rat anti-mouse IL-6 antibody (PE-MP5-20F3, Cat. No. 554401) by using Pharmingen's staining protocol (left panel). To demonstrate specificity of staining, the binding by the PE-MP5-20F3 antibody was blocked by preincubation of the PE-MP5-20F3 antibody with recombinant mouse IL-6 (0.12 µg; Cat. No. 554582; right panel) and by preincubation of the fixed/permeabilized cells with unlabeled MP5-20F3 antibody (5 µg; Cat. No. 554400; right panel) prior to staining. The quadrant markers for the bivariate dot plots were set based on the autofluorescence controls and verified using the recombinant cytokine blocking and unlabeled antibody blocking specificity controls.
Expression of IL-6 by stimulated BALB/c cells. BALB/c bone marrow cells were cultured for 2 weeks in the presence of recombinant mouse GM-CSF (20 ng/ml final concentration; Cat. No. 554586). The cells were washed and stimulated with recombinant mouse IFN-γ (10 ng/ml final concentration; Cat. No. 554587) for 4 hours followed by an overnight stimulation with LPS (1 µg/ml final concentration; Sigma, Cat. #L-8272) and GolgiPlug™ (1 µg/ml final concentration; Cat. No. 555029; aka Brefeldin A). Adherent cells were incubated with 1x trypsin EDTA at 37°C for 10 minutes and gently dislodged by pipetting for cell harvest. Nonspecific binding of cell surface antigens was blocked by incubation of the cells with Fc Block™ (10 µg/ml final concentration; Cat. No. 553142). The cells were fixed , permeabilized then subsequently stained with 0.06 µg of PE-rat anti-mouse IL-6 antibody (PE-MP5-20F3, Cat. No. 554401) by using Pharmingen's staining protocol (left panel). To demonstrate specificity of staining, the binding by the PE-MP5-20F3 antibody was blocked by preincubation of the PE-MP5-20F3 antibody with recombinant mouse IL-6 (0.12 µg; Cat. No. 554582; right panel) and by preincubation of the fixed/permeabilized cells with unlabeled MP5-20F3 antibody (5 µg; Cat. No. 554400; right panel) prior to staining. The quadrant markers for the bivariate dot plots were set based on the autofluorescence controls and verified using the recombinant cytokine blocking and unlabeled antibody blocking specificity controls.
Expression of IL-6 by stimulated BALB/c cells. BALB/c bone marrow cells were cultured for 2 weeks in the presence of recombinant mouse GM-CSF (20 ng/ml final concentration; Cat. No. 554586). The cells were washed and stimulated with recombinant mouse IFN-γ (10 ng/ml final concentration; Cat. No. 554587) for 4 hours followed by an overnight stimulation with LPS (1 µg/ml final concentration; Sigma, Cat. #L-8272) and GolgiPlug™ (1 µg/ml final concentration; Cat. No. 555029; aka Brefeldin A). Adherent cells were incubated with 1x trypsin EDTA at 37°C for 10 minutes and gently dislodged by pipetting for cell harvest. Nonspecific binding of cell surface antigens was blocked by incubation of the cells with Fc Block™ (10 µg/ml final concentration; Cat. No. 553142). The cells were fixed , permeabilized then subsequently stained with 0.06 µg of PE-rat anti-mouse IL-6 antibody (PE-MP5-20F3, Cat. No. 554401) by using Pharmingen's staining protocol (left panel). To demonstrate specificity of staining, the binding by the PE-MP5-20F3 antibody was blocked by preincubation of the PE-MP5-20F3 antibody with recombinant mouse IL-6 (0.12 µg; Cat. No. 554582; right panel) and by preincubation of the fixed/permeabilized cells with unlabeled MP5-20F3 antibody (5 µg; Cat. No. 554400; right panel) prior to staining. The quadrant markers for the bivariate dot plots were set based on the autofluorescence controls and verified using the recombinant cytokine blocking and unlabeled antibody blocking specificity controls.
Expression of IL-6 by stimulated BALB/c cells. BALB/c bone marrow cells were cultured for 2 weeks in the presence of recombinant mouse GM-CSF (20 ng/ml final concentration; Cat. No. 554586). The cells were washed and stimulated with recombinant mouse IFN-γ (10 ng/ml final concentration; Cat. No. 554587) for 4 hours followed by an overnight stimulation with LPS (1 µg/ml final concentration; Sigma, Cat. #L-8272) and GolgiPlug™ (1 µg/ml final concentration; Cat. No. 555029; aka Brefeldin A). Adherent cells were incubated with 1x trypsin EDTA at 37°C for 10 minutes and gently dislodged by pipetting for cell harvest. Nonspecific binding of cell surface antigens was blocked by incubation of the cells with Fc Block™ (10 µg/ml final concentration; Cat. No. 553142). The cells were fixed , permeabilized then subsequently stained with 0.06 µg of PE-rat anti-mouse IL-6 antibody (PE-MP5-20F3, Cat. No. 554401) by using Pharmingen's staining protocol (left panel). To demonstrate specificity of staining, the binding by the PE-MP5-20F3 antibody was blocked by preincubation of the PE-MP5-20F3 antibody with recombinant mouse IL-6 (0.12 µg; Cat. No. 554582; right panel) and by preincubation of the fixed/permeabilized cells with unlabeled MP5-20F3 antibody (5 µg; Cat. No. 554400; right panel) prior to staining. The quadrant markers for the bivariate dot plots were set based on the autofluorescence controls and verified using the recombinant cytokine blocking and unlabeled antibody blocking specificity controls.
Immunofluorescent Staining and Flow Cytometric Analysis: The PE-conjugated MP5-20F3 antibody (Cat. No. 554401) can be used for multicolor immunofluorescent staining and flow cytometric analyses to identify and enumerate IL-6-producing cells within mixed cell populations (see images). For optimal immunofluorescent staining with flow cytometric analysis, this anti-cytokine antibody should be titrated (≤ 0.25 µg mAb/million cells) 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 either of the following: 1) pre-block the conjugated MP5-20F3 antibody with ligand (e.g., recombinant mouse IL-6; Cat No. 554582) prior to staining, or 2) pre-block the fixed/permeabilized cells with unlabeled MP5-20F3 antibody (Cat. No. 554400) prior to staining. The staining technique and use of blocking controls are described in detail by C. Prussin and D. Metcalfe.
A suitable rat IgG1 isotype control for assessing the level of background staining on paraformaldehyde-fixed/saponin-permeabilized mouse or human cells is purified R3-34 (Cat. No. 554685); use at comparable concentrations to antibody of interest (e.g., ≤ 0.25 µg mAb/1 million cells).
Other applications for this clone in additional formats available are:
Neutralization: The NA/LE™ MP5-20F3 antibody (Cat. No. 554398) is useful for neutralization of mouse IL-6 bioactivity.
ELISA Capture: The purified MP5-20F3 antibody (Cat. No. 554401) is useful as a capture antibody for a sandwich ELISA for measuring mouse IL-6 protein levels with the biotinylated detection antibody (Cat. No. 554402) and recombinant mouse IL-6 (Cat. No. 554582) as a standard.
Western Blot: The MP5-20F3 antibody has been reported to be useful for Western blotting. Please note that this application is not tested at BD Biosciences Pharmingen.
The MP5-20F3 monoclonal antibody specifically binds to mouse interleukin-6 (IL-6). The immunogen used to generate the MP5-20F3 hybridoma was recombinant mouse IL-6.
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