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BD Pharmingen™ FITC Rat IgG2a, κ Isotype Control
Clone R35-95 (RUO)
Expression of IL-2 by stimulated CD3+ human PBMC. Human PBMC were stimulated for 6 hours with PMA (Sigma) and calcium ionophore A23187 (Sigma) in the presence of GolgiStop™ (2 mM final concentration; Cat. No. 554714). The PBMC were stained with PE-Cy5-anti-CD3 (PE-CY5 UCHT1, Cat. No 555334), fixed, permeabilized, and subsequently stained with 0.25 mg of FITC-Rat IgG2a, κ anti-human IL-2 antibody (FITC-MQ1-17H12, Cat. No 554565; left panel) or 0.25 mg FITC-R35-95 isotype control immunoglobulin (FITC-R35-95, Cat. No. 554688; right panel) using Pharmingen's staining protocol. To demonstrate specificity of staining, the binding of FITC-MQ1-17H12 was blocked by the preincubation of the conjugated antibody with molar excess of recombinant human IL-2 (Cat. No.554603; data not shown), and by preincubation of the fixed/permeabilized cells with an excess of the unlabelled MQ1-17H12 antibody (Cat. No. 554563; data not shown) prior to staining with the FITC-MQ1-17H12 antibody. The quadrant markers for the bivariate dot plots were set based on the autofluorescence control, and verified with the recombinant cytokine blocking and unlabelled antibody blocking specificity controls.
Expression of IL-2 by stimulated CD3+ human PBMC. Human PBMC were stimulated for 6 hours with PMA (Sigma) and calcium ionophore A23187 (Sigma) in the presence of GolgiStop™ (2 mM final concentration; Cat. No. 554714). The PBMC were stained with PE-Cy5-anti-CD3 (PE-CY5 UCHT1, Cat. No 555334), fixed, permeabilized, and subsequently stained with 0.25 mg of FITC-Rat IgG2a, κ anti-human IL-2 antibody (FITC-MQ1-17H12, Cat. No 554565; left panel) or 0.25 mg FITC-R35-95 isotype control immunoglobulin (FITC-R35-95, Cat. No. 554688; right panel) using Pharmingen's staining protocol. To demonstrate specificity of staining, the binding of FITC-MQ1-17H12 was blocked by the preincubation of the conjugated antibody with molar excess of recombinant human IL-2 (Cat. No.554603; data not shown), and by preincubation of the fixed/permeabilized cells with an excess of the unlabelled MQ1-17H12 antibody (Cat. No. 554563; data not shown) prior to staining with the FITC-MQ1-17H12 antibody. The quadrant markers for the bivariate dot plots were set based on the autofluorescence control, and verified with the recombinant cytokine blocking and unlabelled antibody blocking specificity controls.
Expression of IL-2 by stimulated CD3+ human PBMC. Human PBMC were stimulated for 6 hours with PMA (Sigma) and calcium ionophore A23187 (Sigma) in the presence of GolgiStop™ (2 mM final concentration; Cat. No. 554714). The PBMC were stained with PE-Cy5-anti-CD3 (PE-CY5 UCHT1, Cat. No 555334), fixed, permeabilized, and subsequently stained with 0.25 mg of FITC-Rat IgG2a, κ anti-human IL-2 antibody (FITC-MQ1-17H12, Cat. No 554565; left panel) or 0.25 mg FITC-R35-95 isotype control immunoglobulin (FITC-R35-95, Cat. No. 554688; right panel) using Pharmingen's staining protocol. To demonstrate specificity of staining, the binding of FITC-MQ1-17H12 was blocked by the preincubation of the conjugated antibody with molar excess of recombinant human IL-2 (Cat. No.554603; data not shown), and by preincubation of the fixed/permeabilized cells with an excess of the unlabelled MQ1-17H12 antibody (Cat. No. 554563; data not shown) prior to staining with the FITC-MQ1-17H12 antibody. The quadrant markers for the bivariate dot plots were set based on the autofluorescence control, and verified with the recombinant cytokine blocking and unlabelled antibody blocking specificity controls.
Expression of IL-2 by stimulated CD3+ human PBMC. Human PBMC were stimulated for 6 hours with PMA (Sigma) and calcium ionophore A23187 (Sigma) in the presence of GolgiStop™ (2 mM final concentration; Cat. No. 554714). The PBMC were stained with PE-Cy5-anti-CD3 (PE-CY5 UCHT1, Cat. No 555334), fixed, permeabilized, and subsequently stained with 0.25 mg of FITC-Rat IgG2a, κ anti-human IL-2 antibody (FITC-MQ1-17H12, Cat. No 554565; left panel) or 0.25 mg FITC-R35-95 isotype control immunoglobulin (FITC-R35-95, Cat. No. 554688; right panel) using Pharmingen's staining protocol. To demonstrate specificity of staining, the binding of FITC-MQ1-17H12 was blocked by the preincubation of the conjugated antibody with molar excess of recombinant human IL-2 (Cat. No.554603; data not shown), and by preincubation of the fixed/permeabilized cells with an excess of the unlabelled MQ1-17H12 antibody (Cat. No. 554563; data not shown) prior to staining with the FITC-MQ1-17H12 antibody. The quadrant markers for the bivariate dot plots were set based on the autofluorescence control, and verified with the recombinant cytokine blocking and unlabelled antibody blocking specificity controls.
Product Details
BD Pharmingen™
Rat LOU, also known as Louvain, LOU/C, LOU/M IgG2a, κ
Mouse Pooled Immunoglobulin
Intracellular staining (flow cytometry), Isotype control (Routinely Tested)
0.5 mg/ml
AB_395144
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
Immunofluroescent Staining and Flow Cytometric Analysis: The FITC-R35-95 immunoglobulin (Cat. 554688) is a suitable rat IgG2a κ 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 mg mAb/1 million cells). See image, right panel. For specific methodology, please visit our web site, www.bdbiosciences.com, and go to the protocols section or the chapter on intracellular staining in the Immune Function Handbook. 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.
- 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
Fixation/Permeabilization Solution Kit with BD GolgiPlug™ RUO
Size
250 Tests
Cat No.
555028
Fixation/Permeabilization Kit RUO
Size
250 Tests
Cat No.
554714
553929 Rev. 1
Antibody Details
R35-95
The R35-95 hybridoma was generated by hybridization of Y3 myeloma cells with spleen cells from LOU rats immunized with mouse immunoglobulins. The R35-95 hybridoma produces rat IgG2a, κ immunoglobulin that has no measurable reactivity with mouse immunoglobulins. The R35-95 immunoglobulin was selected as an isotype control following screening for low background binding 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.
553929 Rev. 1
Format Details
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.
FITC
494 nm
518 nm
553929 Rev.1
Citations & References
View product citations for antibody "553929" on CiteAb
Development References (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. (Methodology). View Reference
553929 Rev. 1
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.