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BD Pharmingen™ PE Mouse IgG2b, κ Isotype Control
Clone 27-35 (RUO)
Expression of human IL-8 by stimulated CD14+ human monocytes. Human PBMC were stimulated for 6 hours with LPS (1.0 µg/ml final concentration) in the presence of GolgiStop™ (2 µM final concentration; Cat. No. 554724). The PBMC were harvested, stained with 0.25 µg of FITC-mouse anti-human CD14 monoclonal antibody (FITC-M5E2, Cat. No. 555397), fixed, permeabilized, and subsequently stained with either 0.125 µg of PE-anti-human IL-8 (Cat. No. 554720; left panel), or 0.125 µg of PE-mouse IgG2b (Cat. No. 555058; middle panel), following BD PharMingen staining protocol. The data reflect gating on monocytes, based on forward and side scattered light signals. The quadrant markers for the bivariate dot plot were set based on autofluorescence controls (right panel).
Expression of human IL-8 by stimulated CD14+ human monocytes. Human PBMC were stimulated for 6 hours with LPS (1.0 µg/ml final concentration) in the presence of GolgiStop™ (2 µM final concentration; Cat. No. 554724). The PBMC were harvested, stained with 0.25 µg of FITC-mouse anti-human CD14 monoclonal antibody (FITC-M5E2, Cat. No. 555397), fixed, permeabilized, and subsequently stained with either 0.125 µg of PE-anti-human IL-8 (Cat. No. 554720; left panel), or 0.125 µg of PE-mouse IgG2b (Cat. No. 555058; middle panel), following BD PharMingen staining protocol. The data reflect gating on monocytes, based on forward and side scattered light signals. The quadrant markers for the bivariate dot plot were set based on autofluorescence controls (right panel).
Expression of human IL-8 by stimulated CD14+ human monocytes. Human PBMC were stimulated for 6 hours with LPS (1.0 µg/ml final concentration) in the presence of GolgiStop™ (2 µM final concentration; Cat. No. 554724). The PBMC were harvested, stained with 0.25 µg of FITC-mouse anti-human CD14 monoclonal antibody (FITC-M5E2, Cat. No. 555397), fixed, permeabilized, and subsequently stained with either 0.125 µg of PE-anti-human IL-8 (Cat. No. 554720; left panel), or 0.125 µg of PE-mouse IgG2b (Cat. No. 555058; middle panel), following BD PharMingen staining protocol. The data reflect gating on monocytes, based on forward and side scattered light signals. The quadrant markers for the bivariate dot plot were set based on autofluorescence controls (right panel).
Expression of human IL-8 by stimulated CD14+ human monocytes. Human PBMC were stimulated for 6 hours with LPS (1.0 µg/ml final concentration) in the presence of GolgiStop™ (2 µM final concentration; Cat. No. 554724). The PBMC were harvested, stained with 0.25 µg of FITC-mouse anti-human CD14 monoclonal antibody (FITC-M5E2, Cat. No. 555397), fixed, permeabilized, and subsequently stained with either 0.125 µg of PE-anti-human IL-8 (Cat. No. 554720; left panel), or 0.125 µg of PE-mouse IgG2b (Cat. No. 555058; middle panel), following BD PharMingen staining protocol. The data reflect gating on monocytes, based on forward and side scattered light signals. The quadrant markers for the bivariate dot plot were set based on autofluorescence controls (right panel).
Product Details
BD Pharmingen™
Anti-DNS; Anti-Dansyl; IgG2b kappa; Isotype Control (anti-Dansyl)
Mouse C.SW IgG2b, κ
Dansyl (DNS) Hapten
Flow cytometry, Intracellular staining (flow cytometry), Isotype control (Routinely Tested)
0.2 mg/ml
AB_395678
Aqueous buffered solution containing ≤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 with R-PE under optimum conditions, and unconjugated antibody and free PE were removed. Store undiluted at 4°C and protected from prolonged exposure to light. Do not freeze.
Recommended Assay Procedures
Immunofluorescent Staining and Flow Cytometric Analysis: The PE- and FITC-conjugated 27-35 immunoglobulin (Cat. No. 555057; 555058) are suitable mouse IgG2b isotype controls for assessing the level of background staining on paraformaldehyde fixed/saponin-permeabilized rat 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, please visit the protocols section or chapter on intracellular staining in the Immune Function Handbook, both of which are posted on our web site, www.bdbiosciences.com. The intracellular cytokine staining technique and 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
Fixation/Permeabilization Solution Kit with BD GolgiStop™ RUO
Size
250 Tests
Cat No.
554715
555058 Rev. 2
Antibody Details
27-35
This mouse IgG2b, κ isotype control is a monoclonal antibody, clone 27-35, that is specific for the dansyl (5-[dimethylamino] naphthalene-1-sulfonyl) hapten. The dansyl (DNS) hapten is not expressed on human cells or human cell lines. The 27-35 immunoglobulin was selected as an isotype control following testing that demonstrated low background staining on a variety of mouse and human tissues.
555058 Rev. 2
Format Details
PE
R-Phycoerythrin (PE), is part of the BD family of Phycobiliprotein dyes. This fluorochrome is a multimeric fluorescent phycobiliprotein with excitation maximum (Ex Max) of 496 nm and 566 nm and an emission maximum (Em Max) at 576 nm. PE is designed to be excited by the Blue (488 nm), Green (532 nm) and Yellow-Green (561 nm) lasers and detected using an optical filter centered near 575 nm (e.g., a 575/26-nm bandpass filter). As PE is excited by multiple lasers, this can result in cross-laser excitation and fluorescence spillover on instruments with various combinations of Blue, Green, and Yellow-Green lasers. Please ensure that your instrument’s configurations (lasers and optical filters) are appropriate for this dye.
PE
496 nm, 566 nm
576 nm
555058 Rev.2
Citations & References
View product citations for antibody "555058" on CiteAb
Development References (2)
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BD Biosciences. Techniques for Immune Function Analysis, Application Handbook 1st Edition. 2003. Available: http://www.bdbiosciences.com/pdfs/manuals/02-8100055-21A1rr.pdf 2007, Jan. 25.
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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
555058 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.