Expression of IL-4 by stimulated human lymphocytes. Human peripheral blood mononuclear cells were stimulated for 6 h with Phorbol 12-Myristate 13-Acetate (PMA; Sigma, Cat. No. P-8139) and calcium ionophore A23187 (Sigma, Cat. No. C-9275) in the presence of GolgiStop™ Protein Transport Inhibitor (Cat. No. 554724). The cells were fixed using BD Cytofix™ Fixation Buffer (Cat. No. 554655) and permeabilized with BD Perm/Wash™ Buffer (Cat. No. 554723). The cells were then stained with Pacific Blue™ Mouse Anti-Human CD3 (Cat No. 558117) and PerCP-Cy™5.5 Mouse anti-Human IL-4 antibody (Cat No. 561234, Left Panel) or PerCP-Cy™5.5 Mouse IgG1 κ Isotype Control (Cat No. 550795, Right Panel) by using BD Biosciences Intracellular Cytokine Staining protocol. Two-color flow cytometric dot plots were derived from gated events with the forward and side light-scatter characteristics of intact lymphocytes. Flow cytometry was performed using a BD™ LSR II Flow Cytometer System.
Expression of IL-4 by stimulated human lymphocytes. Human peripheral blood mononuclear cells were stimulated for 6 h with Phorbol 12-Myristate 13-Acetate (PMA; Sigma, Cat. No. P-8139) and calcium ionophore A23187 (Sigma, Cat. No. C-9275) in the presence of GolgiStop™ Protein Transport Inhibitor (Cat. No. 554724). The cells were fixed using BD Cytofix™ Fixation Buffer (Cat. No. 554655) and permeabilized with BD Perm/Wash™ Buffer (Cat. No. 554723). The cells were then stained with Pacific Blue™ Mouse Anti-Human CD3 (Cat No. 558117) and PerCP-Cy™5.5 Mouse anti-Human IL-4 antibody (Cat No. 561234, Left Panel) or PerCP-Cy™5.5 Mouse IgG1 κ Isotype Control (Cat No. 550795, Right Panel) by using BD Biosciences Intracellular Cytokine Staining protocol. Two-color flow cytometric dot plots were derived from gated events with the forward and side light-scatter characteristics of intact lymphocytes. Flow cytometry was performed using a BD™ LSR II Flow Cytometer System.
Product Details
BD Pharmingen™
Interleukin-4; IL4; BCGF-1; BSF-1; Lymphocyte stimulatory factor 1
Human (QC Testing)
Mouse IgG1, κ
Recombinant Human IL-4
Intracellular staining (flow cytometry) (Routinely Tested)
5 µl
AB_10612560
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 PerCP-Cy5.5 under optimum conditions, and unconjugated antibody and free PerCP-Cy5.5 were removed. Storage of PerCP-Cy5.5 conjugates in unoptimized diluent is not recommended and may result in loss of signal intensity.
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.
- PerCP-Cy5.5–labelled antibodies can be used with FITC- and R-PE–labelled reagents in single-laser flow cytometers with no significant spectral overlap of PerCP-Cy5.5, FITC, and R-PE fluorescence.
- PerCP-Cy5.5 is optimized for use with a single argon ion laser emitting 488-nm light. Because of the broad absorption spectrum of the tandem fluorochrome, extra care must be taken when using dual-laser cytometers, which may directly excite both PerCP and Cy5.5™. We recommend the use of cross-beam compensation during data acquisition or software compensation during data analysis.
- This PerCP-conjugated product is sold under license to the following patent: US Patent No. 4,876,190.
- 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 Buffer RUO
Size
100 mL
Cat No.
554655
Perm/Wash Buffer RUO
Size
100 mL
Cat No.
554723
Protein Transport Inhibitor (Containing Monensin) RUO
Size
0.7 mL
Cat No.
554724
PerCP-Cy™5.5 Mouse IgG1 κ Isotype Control RUO
Size
0.1 mg
Cat No.
550795
Pacific Blue™ Mouse Anti-Human CD3 RUO
Size
0.1 mg
Cat No.
558117
561234 Rev. 2
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.
561234 Rev. 2
Format Details
PerCP-Cy5.5
PerCP-Cy5.5 dye is part of the BD blue family of dyes. This tandem fluorochrome is comprised of a fluorescent protein complex (PerCP) with an excitation maximum (Ex Max) of 482 nm and an acceptor dye with an emission maximum (Em Max) at 676 nm. PerCP-Cy5 is designed to be excited by the blue laser (488-nm) and detected using an optical filter centered near 680 nm (e.g., a 695/40 nm bandpass filter). The donor dye can be partially excited by the Violet (405-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.
PerCP-Cy5.5
Blue 488 nm
482 nm
676 nm
561234 Rev.2
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
561234 Rev. 2
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.
