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Alexa Fluor® 647 Rat IgG1, κ Isotype Control
Alexa Fluor® 647 Rat IgG1, κ Isotype Control

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.

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.

Product Details
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BD Pharmingen™
Rat IgG1, κ
Mouse immunoglobulin
Intracellular staining (flow cytometry), Isotype control (Routinely Tested)
5 µl
AB_396908
Aqueous buffered solution containing BSA 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 to Alexa Fluor® 647 under optimum conditions, and unreacted Alexa Fluor® 647 was removed. Store undiluted at 4°C and protected from prolonged exposure to light. Do not freeze.

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

  1. 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).
  2. Since applications vary, each investigator should titrate the reagent to obtain optimal results.
  3. Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
  4. Alexa Fluor® 647 fluorochrome emission is collected at the same instrument settings as for allophycocyanin (APC).
  5. For fluorochrome spectra and suitable instrument settings, please refer to our Multicolor Flow Cytometry web page at www.bdbiosciences.com/colors.
  6. Source of all serum proteins is from USDA inspected abattoirs located in the United States.
  7. 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.
  8. 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.
  9. Alexa Fluor® is a registered trademark of Life Technologies Corporation.
557865 Rev. 3
Antibody Details
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R3-34

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.

557865 Rev. 3
Format Details
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Alexa Fluor™ 647
Alexa Fluor™ 647 Dye is part of the BD red family of dyes. This is a small organic fluorochrome with an excitation maximum (Ex Max) at 653-nm and an emission maximum (Em Max) at 669-nm. Alexa Fluor 647 is designed to be excited by the Red laser (627-640 nm) and detected using an optical filter centered near 520-nm (e.g., a 660/20 nm bandpass filter). Please ensure that your instrument’s configurations (lasers and optical filters) are appropriate for this dye.
Alexa Fluor™ 647
Red 627-640 nm
653 nm
669 nm
557865 Rev.3
Citations & References
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Development References (1)

  1. 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
557865 Rev. 3

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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.