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PE Mouse IgG1, κ Isotype Control
PE Mouse IgG1, κ Isotype Control

Expression of IFN-γ by stimulated human peripheral blood mononuclear cells (PBMC). Human PBMC  were stimulated for 6 hours with PMA (50 ng/ml final concentration; Sigma, Cat. No. P-8139) and calcium ionophore A23187 (Sigma, Cat. No. C-9275), in the presence of GolgiStop™ (2 µM final concentration; Cat. No. 554724). The PBMC were stained with PE-Cy5 anti-CD3 (PE-Cy5-UCHT1, Cat. No. 555334), fixed, permeabilized, and subsequently stained with 0.25 µg of PE-mouse anti-human IFN-γ antibody (PE-4S.B3, Cat. No. 554552, left panel) or with 0.25 µg PE-MOPC-21 immunoglobulin (Cat. No. 554680, right panel) using the BD Pharmingen™ staining protocol. To demonstrate specificity of staining, the binding of PE-4S.B3 antibody was blocked by preincubation of fixed/permeabilized cells with excess unlabelled 4S.B3 antibody (5 µg; Cat. No. 554549). The quadrant markers for the bivariate dot plot were set based on autofluorescence controls and verified using the unlabelled 4S.B3 antibody blocking control.

Expression of IFN-γ by stimulated human peripheral blood mononuclear cells (PBMC). Human PBMC  were stimulated for 6 hours with PMA (50 ng/ml final concentration; Sigma, Cat. No. P-8139) and calcium ionophore A23187 (Sigma, Cat. No. C-9275), in the presence of GolgiStop™ (2 µM final concentration; Cat. No. 554724). The PBMC were stained with PE-Cy5 anti-CD3 (PE-Cy5-UCHT1, Cat. No. 555334), fixed, permeabilized, and subsequently stained with 0.25 µg of PE-mouse anti-human IFN-γ antibody (PE-4S.B3, Cat. No. 554552, left panel) or with 0.25 µg PE-MOPC-21 immunoglobulin (Cat. No. 554680, right panel) using the BD Pharmingen™ staining protocol. To demonstrate specificity of staining, the binding of PE-4S.B3 antibody was blocked by preincubation of fixed/permeabilized cells with excess unlabelled 4S.B3 antibody (5 µg; Cat. No. 554549). The quadrant markers for the bivariate dot plot were set based on autofluorescence controls and verified using the unlabelled 4S.B3 antibody blocking control.

Product Details
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BD Pharmingen™
Mouse IgG1, κ
Flow cytometry, Intracellular staining (flow cytometry), Isotype control (Routinely Tested)
0.2 mg/ml
AB_395506
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-MOPC-21 immunoglobulins (Cat. No. 554680) is a suitable mouse IgG1κ isotype control for assessing the level of background staining on paraformaldehyde fixed/saponin-permeabilized mouse 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, visit the protocols section of our website, or the chapter on intracellular staining in the Immune Function Handbook, which is posted on our web site at www.bdbiosciences.com. The intracellular cytokine staining technique and the use of blocking controls are described in detail by C. Prussin and D. Metcalfe.

Product Notices

  1. Since applications vary, each investigator should titrate the reagent to obtain optimal results.
  2. Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
  3. For fluorochrome spectra and suitable instrument settings, please refer to our Multicolor Flow Cytometry web page at www.bdbiosciences.com/colors.
  4. 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.
554680 Rev. 2
Antibody Details
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MOPC-21

The MOPC-21 immunoglobulin is a mouse myeloma protein. The MOPC-21 immunoglobulin was selected as an isotype control following screening for low background on a variety of mouse and human tissues.

554680 Rev. 2
Format Details
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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.
altImg
PE
Yellow-Green 488 nm, 532 nm, 561 nm
496 nm, 566 nm
576 nm
554680 Rev.2
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. (Biology: Flow cytometry). View Reference
554680 Rev. 2

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For Research Use Only. Not for use in diagnostic or therapeutic procedures.