Flow cytometric analysis for IL-4 in stimulated human peripheral blood mononuclear cells (PBMC). Human PBMC were stimulated for at least 6 hours with 50 ng/mL PMA (Sigma-Aldrich Cat. No. P-8139) and 250 ng/mL calcium ionophore A23187 (Sigma-Aldrich Cat. No. C-9275) in the presence of 2 µM BD GolgiStop™ (Cat. No. 554724). Cells were then fixed and permeabilized using BD Cytofix/Cytoperm™ (Cat. No. 554714) followed by staining with either a PE-Cy™7 Mouse IgG1, κ isotype control (left panel) or with the PE-Cy™7 Mouse Anti-Human IL-4 antibody (right panel). Dot plots were derived from gated events based on light scattering characteristics for lymphocytes. Flow cytometry was performed on a BD™ LSR II flow cytometry system.
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Flow cytometric analysis for IL-4 in stimulated human peripheral blood mononuclear cells (PBMC). Human PBMC were stimulated for at least 6 hours with 50 ng/mL PMA (Sigma-Aldrich Cat. No. P-8139) and 250 ng/mL calcium ionophore A23187 (Sigma-Aldrich Cat. No. C-9275) in the presence of 2 µM BD GolgiStop™ (Cat. No. 554724). Cells were then fixed and permeabilized using BD Cytofix/Cytoperm™ (Cat. No. 554714) followed by staining with either a PE-Cy™7 Mouse IgG1, κ isotype control (left panel) or with the PE-Cy™7 Mouse Anti-Human IL-4 antibody (right panel). Dot plots were derived from gated events based on light scattering characteristics for lymphocytes. Flow cytometry was performed on a BD™ LSR II flow cytometry system.
Product Details
BD Pharmingen™
Human (QC Testing)
Mouse IgG1, κ
Recombinant Human IL-4
Intracellular staining (flow cytometry) (Routinely Tested)
5 µl
AB_1727547
Aqueous buffered solution containing BSA and ≤0.09% sodium azide.
RUO
Preparation And Storage
Store undiluted at 4°C and protected from prolonged exposure to light. Do not freeze. The monoclonal antibody was purified from tissue culture supernatant or ascites by affinity chromatography. The antibody was conjugated with PE-Cy7 under optimum conditions, and unconjugated antibody and free PE-Cy7 were removed.
Recommended Assay Procedures
Flow cytometry: The 8D4-8 antibody is useful for immunofluorescent staining and flow cytometric analysis to identify and enumerate IL-4 producing cells within mixed cell populations. A useful control investigators may consider using for demonstrating specificity of staining, is to pre-block with one of the following reagents: (1) recombinant human IL-4 (Cat. No. 554605) or (2) unlabeled 8D4-8 antibody (Cat. No. 554515), prior to staining.
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).
- An isotype control should be used at the same concentration as the antibody of interest.
- 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.
- Source of all serum proteins is from USDA inspected abattoirs located in the United States.
- Please observe the following precautions: Absorption of visible light can significantly alter the energy transfer occurring in any tandem fluorochrome conjugate; therefore, we recommend that special precautions be taken (such as wrapping vials, tubes, or racks in aluminum foil) to prevent exposure of conjugated reagents, including cells stained with those reagents, to room illumination.
- PE-Cy7 is a tandem fluorochrome composed of R-phycoerythrin (PE), which is excited by 488-nm light and serves as an energy donor, coupled to the cyanine dye Cy7, which acts as an energy acceptor and fluoresces maximally at 780 nm. PE-Cy7 tandem fluorochrome emission is collected in a detector for fluorescence wavelengths of 750 nm and higher. Although every effort is made to minimize the lot-to-lot variation in the efficiency of the fluorochrome energy transfer, differences in the residual emission from PE may be observed. Therefore, we recommend that individual compensation controls be performed for every PE-Cy7 conjugate. PE-Cy7 is optimized for use with a single argon ion laser emitting 488-nm light, and there is no significant overlap between PE-Cy7 and FITC emission spectra. When using dual-laser cytometers, which may directly excite both PE and Cy7, we recommend the use of cross-beam compensation during data acquisition or software compensation during data analysis.
- Warning: Some APC-Cy7 and PE-Cy7 conjugates show changes in their emission spectrum with prolonged exposure to formaldehyde. If you are unable to analyze fixed samples within four hours, we recommend that you use BD™ Stabilizing Fixative (Cat. No. 338036).
- Cy is a trademark of Amersham Biosciences Limited.
- For fluorochrome spectra and suitable instrument settings, please refer to our Multicolor Flow Cytometry web page at www.bdbiosciences.com/colors.
- Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
Companion Products
Fixation/Permeabilization Kit RUO
Size
250 Tests
Cat No.
554714
Protein Transport Inhibitor (Containing Monensin) RUO
Size
0.7 mL
Cat No.
554724
PE-Cy™7 Mouse IgG1 κ Isotype Control RUO
Size
100 Tests
Cat No.
557646
HiCK-2 Human Cytokine Positive Control Cells RUO
Size
1 mL
Cat No.
555062
Recombinant Human IL-4 RUO
Size
5 µg
Cat No.
554605
Purified Mouse Anti-Human IL-4 RUO
Size
0.5 mg
Cat No.
554515
560672 Rev. 3
Antibody Details
8D4-8
The 8D4-8 monoclonal antibody reacts with human interleukin-4 (IL-4). The immunogen used to raise the 8D4-8 hybridoma was recombinant human IL-4. The 8D4-8 antibody binds to an epitope that is different than the epitope recognized by the MP4-25D2 antibody (Cat. No. 554485).
Clone 8D4-8 displays an increased amount of non-specific binding to dead cells when compared to the clone MP4-25D2. It is recommended to use a fixable viability dye in conjunction with this clone.
560672 Rev. 3
Format Details
PE-Cy7
PE-Cy7 dye is a part of the BD PE family of dyes. This tandem fluorochrome is comprised of a R-Phycoerythrin (PE) donor that has excitation maxima (Ex Max) of 496-nm and 566-nm and an acceptor dye, Cy™7, with an emission maximum (Em Max) at 781-nm. PE can be excited by the Blue (488-nm), Green (532-nm) and yellow-green (561-nm) lasers and detected using an optical filter centered near 781 nm (e.g., a 760/60-nm bandpass filter). The donor dye can be excited by the Blue (488-nm), Green (532-nm) and yellow-green (561-nm) lasers and the acceptor dye can be excited by the Red (627–640-nm) laser resulting in cross-laser excitation and fluorescence spillover. Please ensure that your instrument’s configurations (lasers and optical filters) are appropriate for this dye.
PE-Cy7
Yellow-Green 488 nm, 532 nm, 561 nm
496 nm, 566 nm
781 nm
560672 Rev.3
Citations & References
Development References (2)
-
Bird C, Wadhwa M, Thorpe R. Development of immunoassays for human interleukin 3 and interleukin 4, some of which discriminate between different recombinant DNA-derived molecules. Cytokine. 1991; 3(6):562-567. (Biology). View Reference
-
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: Flow cytometry). View Reference
560672 Rev. 3
Please refer to Support Documents for Quality Certificates
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.
