Skip to main content Skip to navigation
Alexa Fluor® 488 Rat IgG1 κ Isotype Control
Alexa Fluor® 488 Rat IgG1 κ Isotype Control

Expression of TNF by stimulated CD4+ and CD4- BALB/c spleen cells. Splenocytes from BALB/c mice were stimulated for 4 hours with PMA (5 ng/ml, Sigma, Cat. No. P-8139) and Ionomycin (500 ng, Sigma, I-0634) in the presence of Brefeldin A (GolgiPlug, Cat. No. 555029). Cells were harvested, fixed, permeabilized and stained with PE-conjugated rat anti-mouse CD4 (PE-RM4-5, Cat. No. 553048) and either rat anti-mouse TNF antibody (Alexa Fluor® 488-MP6-XT22, Cat. No. 557719), (left panel) or immunoglobulin isotype control (Alexa Fluor® 488-R3-34, Cat. No. 557720), (right panel) by using the BD Pharmingen staining protocol. Dot plots were derived from gated events with the forward and side light scatter characteristics of lymphocytes. The quadrant markers for the bivariate dot plots were set based on the autofluorescence and isotype controls.

Expression of TNF by stimulated CD4+ and CD4- BALB/c spleen cells. Splenocytes from BALB/c mice were stimulated for 4 hours with PMA (5 ng/ml, Sigma, Cat. No. P-8139) and Ionomycin (500 ng, Sigma, I-0634) in the presence of Brefeldin A (GolgiPlug, Cat. No. 555029). Cells were harvested, fixed, permeabilized and stained with PE-conjugated rat anti-mouse CD4 (PE-RM4-5, Cat. No. 553048) and either rat anti-mouse TNF antibody (Alexa Fluor® 488-MP6-XT22, Cat. No. 557719), (left panel) or immunoglobulin isotype control (Alexa Fluor® 488-R3-34, Cat. No. 557720), (right panel) by using the BD Pharmingen staining protocol. Dot plots were derived from gated events with the forward and side light scatter characteristics of lymphocytes. The quadrant markers for the bivariate dot plots were set based on the autofluorescence and isotype controls.

Product Details
Down Arrow Up Arrow


BD Pharmingen™
Rat IgG1, κ
Mouse immunoglobulin
Flow cytometry, Intracellular staining (flow cytometry), Isotype control (Routinely Tested)
0.2 mg/ml
AB_396829
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 to Alexa Fluor® 488 under optimum conditions, and unreacted Alexa Fluor® 488 was removed. Store undiluted at 4°C and protected from prolonged exposure to light. Do not freeze.

Recommended Assay Procedures

Immunofluorescent Staining for Intracellular Cytokines: The FITC-, PE-, APC-, PE-Cy7-, Alexa Fluor® 488- and Alexa Fluor® 647-conjugated-R3-34 immunoglobulin (Cat. No. 554684, 554685, 554686, 557645, 557720, and 557731) is a suitable rat 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. The intracellular cytokine staining technique and 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. Alexa Fluor® 488 fluorochrome emission is collected at the same instrument settings as for fluorescein isothiocyanate (FITC).
  4. For fluorochrome spectra and suitable instrument settings, please refer to our Multicolor Flow Cytometry web page at www.bdbiosciences.com/colors.
  5. The Alexa Fluor®, Pacific Blue™, and Cascade Blue® dye antibody conjugates in this product are sold under license from Molecular Probes, Inc. for research use only, excluding use in combination with microarrays, or as analyte specific reagents. The Alexa Fluor® dyes (except for Alexa Fluor® 430), Pacific Blue™ dye, and Cascade Blue® dye are covered by pending and issued patents.
  6. 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.
  7. Alexa Fluor® is a registered trademark of Molecular Probes, Inc., Eugene, OR.
557720 Rev. 2
Antibody Details
Down Arrow Up Arrow
R3-34

Following immunization of a rat with mouse immunoglobulin (Ig), the Ig from the R3-34 hybridoma was identified as a non-reactive clone. The R3-34 immunoglobulin was selected as an Ig isotype control following screening for low background staining on a variety of mouse and human cells and 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.

557720 Rev. 2
Format Details
Down Arrow Up Arrow
Alexa Fluor™ 488
Alexa Fluor™ 488 Dye 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 517-nm. Alexa Fluor™ 488 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.
Alexa Fluor™ 488
Blue 488 nm
494 nm
517 nm
557720 Rev.2
Citations & References
Down Arrow Up Arrow

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. (Methodology). View Reference
557720 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.