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Expression of IL-4 by stimulated human peripheral blood mononuclear cells. Human peripheral blood mononuclear cells were stimulated with plate-bound anti-human CD3 (HIT3a, 10 µg/ml, Cat. No. 555336) and soluble anti-CD28 (CD28.2, 2 µg/ml, Cat. No. 555725) antibody in the presence of recombinant human IL-2 (10 ng/ml, Cat. No. 554603) and IL-4 (25 ng/ml, Cat. No. 554605) 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 hours with PMA (5 ng/ml, Sigma, Cat. No. P-8139) and ionomycin (500 ng, Sigma, I-0634) in the presence of Brefeldin A (GolgiPlug, Cat. No. 555029). Following incubation the cells were harvested and stained with PE-anti-human CD4 (Cat. No. 555347) and either rat anti-human IL-4 (Alexa Fluor® 647-MP4-25D2) (left panel) or immunoglobulin isotype control (Alexa Fluor® 647-R3-34, Cat. No. 557865) (right panel) by using Pharmingen's staining protocol. To demonstrate specificity of staining the binding of Alexa Fluor® 647 was blocked by preincubation of the fixed/permeabilized cells with an excess of unlabeled MP4-25D2 antibody (5 µg, Cat. No. 554482, data not shown) prior to staining. The quadrant markers for the bivariate dot plots were set based on the autofluorescence and isotype controls.
BD Pharmingen™ Alexa Fluor® 647 Rat 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 for Intracellular Cytokines: The FITC-, PE-, APC-, PE-Cy7-, Alexa Fluor® 488- and Alexa Fluor® 647-conjugated-R3-34 immunoglobulin (Cat. No. 554684, 554685, 554686, 557645, 557856, and 557865) is a suitable rat IgG1 isotype control for assessing the level of background staining on paraformaldehyde-fixed/saponin-permeabilized mouse or human cells for flow cytometric analysis. The intracellular cytokine staining technique and use of blocking controls are described in detail by C. Prussin and D. Metcalfe.
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).
- 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® 647 fluorochrome emission is collected at the same instrument settings as for allophycocyanin (APC).
- For fluorochrome spectra and suitable instrument settings, please refer to our Multicolor Flow Cytometry web page at www.bdbiosciences.com/colors.
- Source of all serum proteins is from USDA inspected abattoirs located in the United States.
- 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.
- The Alexa Fluor®, Pacific Blue™, and Cascade Blue® dye antibody conjugates in this product are sold under license from Molecular Probes, Inc. for research use only, excluding use in combination with microarrays, or as analyte specific reagents. The Alexa Fluor® dyes (except for Alexa Fluor® 430), Pacific Blue™ dye, and Cascade Blue® dye are covered by pending and issued patents.
- Alexa Fluor® is a registered trademark of Life Technologies Corporation.
Following immunization of a rat with mouse immunoglobulin (Ig), the Ig from the R3-34 hybridoma was identified as a non-reactive clone. The R3-34 immunoglobulin was selected as an Ig isotype control following screening for low background staining on a variety of mouse and human cells and tissues.
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 (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. (Methodology). View Reference
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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.