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Expression of IFN-γ by stimulated human peripheral blood mononuclear cells (PBMC). Human PBMC were stimulated for 6 hours with PMA (50 ng/ml final concentration; Sigma, Cat. No. P-8139) and calcium ionophore A23187 (Sigma, Cat. No. C-9275), in the presence of GolgiStop™ (2 µM final concentration; Cat. No. 554724). The PBMC were stained with PE-Cy5 anti-CD3 (PE-Cy5-UCHT1, Cat. No. 555334), fixed, permeabilized, and subsequently stained with 0.25 µg of PE-mouse anti-human IFN-γ antibody (PE-4S.B3, Cat. No. 554552, left panel) or with 0.25 µg PE-MOPC-21 immunoglobulin (Cat. No. 554680, right panel) using the BD Pharmingen™ staining protocol. To demonstrate specificity of staining, the binding of PE-4S.B3 antibody was blocked by preincubation of fixed/permeabilized cells with excess unlabelled 4S.B3 antibody (5 µg; Cat. No. 554549). The quadrant markers for the bivariate dot plot were set based on autofluorescence controls and verified using the unlabelled 4S.B3 antibody blocking control.
BD Pharmingen™ PE Mouse IgG1, κ Isotype Control
Regulatory Status Legend
Any use of products other than the permitted use without the express written authorization of Becton, Dickinson and Company is strictly prohibited.
Preparation And Storage
Recommended Assay Procedures
Immunofluorescent Staining and Flow Cytometric Analysis: The PE-MOPC-21 immunoglobulins (Cat. No. 554680) is a suitable mouse IgG1κ isotype control for assessing the level of background staining on paraformaldehyde fixed/saponin-permeabilized mouse or human cells for flow cytometric analysis. Use at comparable concentrations to antibody of interest (e.g., ≤ 0.5 µg mAb/1 million cells), (see image, right panel). For specific methodology, visit the protocols section of our website, or the chapter on intracellular staining in the Immune Function Handbook, which is posted on our web site at www.bdbiosciences.com. The intracellular cytokine staining technique and the use of blocking controls are described in detail by C. Prussin and D. Metcalfe.
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.
- 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.
Companion Products
The MOPC-21 immunoglobulin is a mouse myeloma protein. The MOPC-21 immunoglobulin was selected as an isotype control following screening for low background on a variety of mouse and human tissues.
Development References (1)
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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. (Biology: Flow cytometry). View Reference
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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.