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PE Rat Anti-Mouse IL-3
PE Rat Anti-Mouse IL-3

Expression of IL-3 by stimulated CD4+ Balb/c spleen cells. Purified splenic CD4+ cells from 6 month old BALB/c mice were stimulated with plate-bound anti-CD3 (25 µg/ml final concentration; 145-2C11, Cat. No. 553057) and soluble anti-mouse CD28 (2 µg /ml final concentration; clone 37.51, Cat. No. 553294) for 2 days in culture  together with recombinant mouse IL-2 (10 ng/ml final concentration; Cat. No. 550069) and recombinant mouse IL-4 (0.5 ng/ml final concentration; Cat. No. 550067), followed by a 3 day incubation with only recombinant mouse IL-2 and recombinant mouse IL-4. This was followed by a 5 hour stimulation with plate-bound anti-CD3 (25 µg/ml final concentration) and anti-mouse CD28 (2 µg/ml final concentration) in the presence of GolgiStop™ (2 µM final concentration; Cat. No. 554724). The cells were harvested, stained with 0.06 µg of FITC-conjugated rat anti-mouse CD4 (FITC-RM4-5, Cat. No. 553047), fixed, permeabilized, and subsequently stained with 0.12 µg of PE-conjugated rat anti-mouse IL-3 antibody (PE-MP2-8F8, Cat. No. 554383) by using the BD Pharmingen staining protocol (left panel). To demonstrate specificity of staining, the binding by PE-MP2-8F8 was blocked by each of the following: 1) preincubation of the conjugated antibody with molar excess of recombinant mouse IL-3 (0.12 µg, Cat. No. 554579; center panel) and by 2)   preincubation of the fixed/permeabilized cells with excess unlabeled MP2-8F8 mouse antibody (3 µg; Cat. No. 554380; right panel) prior to staining with the PE-MP2-8F8. The quadrant markers for the bivariate dot plots were set based on the autofluorescence controls and verified using the recombinant cytokine blocking and unlabeled antibody blocking specificity controls.

Expression of IL-3 by stimulated CD4+ Balb/c spleen cells. Purified splenic CD4+ cells from 6 month old BALB/c mice were stimulated with plate-bound anti-CD3 (25 µg/ml final concentration; 145-2C11, Cat. No. 553057) and soluble anti-mouse CD28 (2 µg /ml final concentration; clone 37.51, Cat. No. 553294) for 2 days in culture  together with recombinant mouse IL-2 (10 ng/ml final concentration; Cat. No. 550069) and recombinant mouse IL-4 (0.5 ng/ml final concentration; Cat. No. 550067), followed by a 3 day incubation with only recombinant mouse IL-2 and recombinant mouse IL-4. This was followed by a 5 hour stimulation with plate-bound anti-CD3 (25 µg/ml final concentration) and anti-mouse CD28 (2 µg/ml final concentration) in the presence of GolgiStop™ (2 µM final concentration; Cat. No. 554724). The cells were harvested, stained with 0.06 µg of FITC-conjugated rat anti-mouse CD4 (FITC-RM4-5, Cat. No. 553047), fixed, permeabilized, and subsequently stained with 0.12 µg of PE-conjugated rat anti-mouse IL-3 antibody (PE-MP2-8F8, Cat. No. 554383) by using the BD Pharmingen staining protocol (left panel). To demonstrate specificity of staining, the binding by PE-MP2-8F8 was blocked by each of the following: 1) preincubation of the conjugated antibody with molar excess of recombinant mouse IL-3 (0.12 µg, Cat. No. 554579; center panel) and by 2)   preincubation of the fixed/permeabilized cells with excess unlabeled MP2-8F8 mouse antibody (3 µg; Cat. No. 554380; right panel) prior to staining with the PE-MP2-8F8. The quadrant markers for the bivariate dot plots were set based on the autofluorescence controls and verified using the recombinant cytokine blocking and unlabeled antibody blocking specificity controls.

Product Details
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BD Pharmingen™
Mouse (QC Testing)
Rat IgG1
Recombinant mouse IL-3
Intracellular staining (flow cytometry) (Routinely Tested)
0.2 mg/ml
AB_395358
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 MP2-8F8 antibody is useful for immunofluorescent staining and flow cytometric analysis to identify and enumerate IL-3 producing cells within mixed cell populations. PE-conjugated MP2-8F8 antibody (Cat. No. 554383) is especially suitable for these studies (see Figure, left panel). 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 MP2-8F8 antibody with a molar excess of ligand (e.g., recombinant mouse IL-3; Cat No. 554579) prior to staining, or 2) pre-block the fixed/permeabilized cells with unlabeled MP2-8F8 antibody (Cat. No. 554380, 554381) prior to staining. The staining technique and use of blocking controls are described in detail by C. Prussin and D. Metcalfe. A suitable rat IgG1 isotype control for assessing the level of background staining on paraformaldehyde-fixed/saponin-permeabilized mouse or human cells is PE-R3-34 (Cat. No. 554685); use at comparable concentrations to antibody of interest (e.g., ≤ 0.25 µg mAb/1 million cells).

Neutralization: The NA/LE™ MP2-8F8 antibody (Cat. No. 554379) is useful for neutralization of mouse IL-3 bioactivity.

ELISA Capture: The purified MP2-8F8 antibody (Cat. No. 554380) is useful as a capture antibody for a sandwich ELISA for measuring mouse IL-3 protein levels. For specific methodology, please visit the protocols section or chapter on ELISA in the Immune Function Handbook, both of which are posted on our web site, www.bdbiosciences.com. For testing mouse IL-3 in complex biological fluids such as serum or plasma, our mouse IL-3 specific OptEIA™ sandwich ELISA set is recommended (Cat. No. 555228).

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.
554383 Rev. 1
Antibody Details
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MP2-8F8

The MP2-8F8 antibody reacts with mouse interleukin-3 (IL-3). The immunogen used to generate the MP2-8F8 hybridoma was COS-expressed recombinant mouse IL-3.

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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 (6)

  1. Abrams J. Immunoenzymetric assay of mouse and human cytokines using NIP-labeled anti-cytokine antibodies. Curr Protoc Immunol. 2001; 1:6.20-6.21. (Clone-specific: ELISA). View Reference
  2. Abrams JS, Pearce MK.. Development of rat anti-mouse interleukin 3 monoclonal antibodies which neutralize bioactivity in vitro. J Immunol. 1988; 140(1):131-137. (Clone-specific: ELISA, Neutralization). View Reference
  3. Abrams JS, Roncarolo MG, Yssel H, Andersson U, Gleich GJ, Silver JE. Strategies of anti-cytokine monoclonal antibody development: immunoassay of IL-10 and IL-5 in clinical samples. Immunol Rev. 1992; 127:5-24. (Clone-specific: ELISA, Neutralization). View Reference
  4. Cockayne DA, Muchamuel T, Grimaldi JC, et al. Mice deficient for the ecto-nicotinamide adenine dinucleotide glycohydrolase CD38 exhibit altered humoral immune responses. Blood. 1998; 92(4):1324-1333. (Clone-specific: ELISA, 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: IC/FCM Block). View Reference
  6. Sander B, Hoiden I, Andersson U, Moller E, Abrams JS. Similar frequencies and kinetics of cytokine producing cells in murine peripheral blood and spleen. Cytokine detection by immunoassay and intracellular immunostaining. J Immunol Methods. 1993; 166(2):201-214. (Clone-specific: ELISA). View Reference
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For Research Use Only. Not for use in diagnostic or therapeutic procedures.