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FITC Mouse IgG1, κ Isotype Control
FITC 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 ionophone A23187 (250 ng/ml final concentration; Sigma, Cat. No. C-9275) in  the presence of GolgiStop™ (2 µM final concentration; Cat. No. 554724). The PBMC were stained with PE-Cy5 (formerly referred to as BD-Cy-Chrome™) - anti-CD3 (Cy-Chrome™ - UCHT1, Cat. No. 555334), fixed, permeabilized, and subsequently stained with 0.25 µg of FITC-mouse anti-human IFN-γ antibody (FITC-4S.B3, Cat. No. 554551; left panel) or FITC-MOPC-21 immunoglobulin (0.25 µg; Cat. No. 554679; right panel) by using the BD Bioscience staining protocol. To demonstrate specificity of staining,  the binding of FITC-4S.B3 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 ionophone A23187 (250 ng/ml final concentration; Sigma, Cat. No. C-9275) in  the presence of GolgiStop™ (2 µM final concentration; Cat. No. 554724). The PBMC were stained with PE-Cy5 (formerly referred to as BD-Cy-Chrome™) - anti-CD3 (Cy-Chrome™ - UCHT1, Cat. No. 555334), fixed, permeabilized, and subsequently stained with 0.25 µg of FITC-mouse anti-human IFN-γ antibody (FITC-4S.B3, Cat. No. 554551; left panel) or FITC-MOPC-21 immunoglobulin (0.25 µg; Cat. No. 554679; right panel) by using the BD Bioscience staining protocol. To demonstrate specificity of staining,  the binding of FITC-4S.B3 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)
0.5 mg/ml
AB_395505
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 FITC under optimum conditions, and unreacted FITC was 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 FITC-MOPC-21 immunoglobulin (Cat. No. 554679) is a suitable mouse IgG1 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) (figure right panel).

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. 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.
554679 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.

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.

554679 Rev. 2
Format Details
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FITC
Fluorescein (FITC) is part of the BD blue family of dyes. This is a small organic fluorochrome with an excitation maximum (Ex Max) at 494-nm and an emission maximum (Em Max) at 518-nm. FITC is designed to be excited by the Blue laser (488-nm) and detected using an optical filter centered near 520 nm (e.g., a 530/30-nm bandpass filter). Please ensure that your instrument’s configurations (lasers and optical filters) are appropriate for this dye.
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FITC
Blue 488 nm
494 nm
518 nm
554679 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
554679 Rev. 2

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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.

Refer to manufacturer's instructions for use and related User Manuals and Technical Data Sheets before using this product 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.