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PE Hamster Anti-Mouse IL-1α
PE Hamster Anti-Mouse IL-1α

Expression of IL-1α by activated mouse peritoneal macrophages. Thioglycolate-elicited BALB/c mouse peritoneal macrophages were primed for 2 hour with rmIFN-γ (10 µg/ml, Cat. No. 554587) and stimulated overnight with LPS (1 µg/ml, Sigma Cat. No. L-8274) in the presence of BD GolgiPlug™ (containing Brefeldin A, Cat. No. 555029). The activated cells were harvested, fixed, permeabilized and stained with PE-conjugated anti-mouse IL-1a antibody (PE-ALF-161, Cat. No. 559810; left panel) following BD Biosciences Pharmingen's intracellular staining protocol. To demonstrate specificity of staining, the binding of PE- ALF-161 antibody was blocked by preincubation of the conjugated antibody with recombinant mouse IL-1α (0.25 µg, Cat. No. 551778; middle panel) or by preincubation of the fixed/ permeabilized cells with unlabelled ALF-161 antibody (5.0 µg; Cat. No. 550604; right panel) prior to staining with the PE- ALF-161 antibody. The quadrant markers for the bivariate dot plots were set based on the autofluorescence controls and verified using the recombinant cytokine blocking and unlabelled antibody blocking specificity controls.

Expression of IL-1α by activated mouse peritoneal macrophages. Thioglycolate-elicited BALB/c mouse peritoneal macrophages were primed for 2 hour with rmIFN-γ (10 µg/ml, Cat. No. 554587) and stimulated overnight with LPS (1 µg/ml, Sigma Cat. No. L-8274) in the presence of BD GolgiPlug™ (containing Brefeldin A, Cat. No. 555029). The activated cells were harvested, fixed, permeabilized and stained with PE-conjugated anti-mouse IL-1a antibody (PE-ALF-161, Cat. No. 559810; left panel) following BD Biosciences Pharmingen's intracellular staining protocol. To demonstrate specificity of staining, the binding of PE- ALF-161 antibody was blocked by preincubation of the conjugated antibody with recombinant mouse IL-1α (0.25 µg, Cat. No. 551778; middle panel) or by preincubation of the fixed/ permeabilized cells with unlabelled ALF-161 antibody (5.0 µg; Cat. No. 550604; right panel) prior to staining with the PE- ALF-161 antibody. The quadrant markers for the bivariate dot plots were set based on the autofluorescence controls and verified using the recombinant cytokine blocking and unlabelled antibody blocking specificity controls.

Product Details
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BD Pharmingen™
Mouse (QC Testing)
Armenian Hamster IgG, λ
Mouse IL-1α recombinant protein
Intracellular staining (flow cytometry) (Routinely Tested)
0.2 mg/ml
AB_397339
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 Cytometry Analysis: The PE-conjugated ALF-161 antibody can be used for multicolor immunofluorescent staining and flow cytometric analyses to identify and enumerate IL-1α-producing cells within mixed cell populations (see figure). For optimal immunofluorescent staining with flow cytometric analysis, this anti-cytokine antibody should be titrated (≤ 0.25 µg mAb/million cells). 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.

A useful control for demonstrating specificity of staining is either of the following: 1) pre-block the conjugated ALF-161 antibody with its cognate ligand (e.g., recombinant mouse IL-1α) prior to staining, or 2) pre-block the fixed/permeabilized cells with unlabelled ALF-161 antibody (Cat. No. 550604) prior to staining. The intracellular cytokine staining technique and use of blocking controls are described in detail by C. Prussin and D. Metcalfe. A suitable Armenian hamster IgG isotype control for assessing the level of background staining on paraformaldehyde-fixed/saponin-permeabilized mouse or human cells is PE-G235-2356 (Cat. No. 554711); use at comparable concentrations to antibody of interest (e.g., ≤ 0.25 µg mAb/1 million cells).

ELISA: The ALF-161 antibody is useful as a capture antibody in a sandwich ELISA. Cat. No. 550604 is recommended for this application.

Neutralization: The ALF-161 antibody is useful for neutralization of mouse IL-1α bioactivity.

Western Blot: The purified ALF-161 antibody has been found useful for Western blotting. Please note that this application is not routinely tested at BD Biosciences Pharmingen.

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.
559810 Rev. 1
Antibody Details
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ALF-161

This antibody recognizes the precursor, secreted and membrane-associated forms of mouse interleukin-1α (IL-1α) protein. No cross-reactivity was detected with mouse IL-1β. This antibody does not recognize human IL-1α or IL-1β. The cross-reactivity of this antibody with IL-1α from other species has not been tested. The immunogen used to generate this ALF-161 hybridoma was purified, recombinant mouse IL-1α protein. This is a neutralizing antibody.

559810 Rev. 1
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
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Citations & References
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Development References (5)

  1. Chang MJ, Modzelewski RA, Russell DM, Johnson CS. Interleukin 1 alpha and gamma-interferon induction of nitric oxide production from murine tumor-derived endothelial cells. Cancer Res. 1996; 56(4):886-891. (Biology). View Reference
  2. Fuhlbrigge RC, Sheehan KC, Schreiber RD, Chaplin DD, Unanue ER. Monoclonal antibodies to murine IL-1 alpha. Production, characterization, and inhibition of membrane-associated IL-1 activity. J Immunol. 1988; 141(8):2643-2650. (Clone-specific: Neutralization). View Reference
  3. Kitamura T, Takaku F, Miyajima A. IL-1 up-regulates the expression of cytokine receptors on a factor-dependent human hemopoietic cell line, TF-1. Int Immunol. 1991; 3(6):571-577. (Clone-specific: Neutralization). View Reference
  4. Kitamura T, Tange T, Terasawa T, et al. Establishment and characterization of a unique human cell line that proliferates dependently on GM-CSF, IL-3, or erythropoietin. J Cell Physiol. 1989; 140(2):323-334. (Clone-specific: Neutralization). View Reference
  5. 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
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For Research Use Only. Not for use in diagnostic or therapeutic procedures.