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PE Rat Anti-Mouse GM-CSF
PE Rat Anti-Mouse GM-CSF

Expression of GM-CSF 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; clone 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 (1 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 hours stimulation with plate-bound anti-CD3 (25 µg/ml final concentration) and anti-mouse CD28 (2 µg/ml final concentration) in the presence of BD GolgiStop™ (2 µM final concentration; Cat. No. 554724). The cells were then stained with 0.06 µg of FITC rat anti-mouse CD4 (FITC-RM4-5, Cat. No. 553047) fixed, permeabilized, and subsequently stained with 0.12 µg of PE rat anti-mouse GM-CSF antibody by using the Pharmingen staining protocol (left panel). To demonstrate specificity of staining, the binding of PE-MP1-22E9 was blocked by the preincubation of the conjugated antibody with recombinant mouse GM-CSF (1 µg, Cat. No. 554586; center panel), and by preincubation of the fixed/permeabilized cells with the unlabelled MP1-22E9 mouse antibody (10 µg, Cat. No. 556916; right panel). The quadrant markers for the bivariate dot plots were set based on the autofluorescence control, and verified with the recombinant cytokine blocking (center panel) and unlabelled antibody blocking (right panel) specificity controls.

Expression of GM-CSF 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; clone 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 (1 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 hours stimulation with plate-bound anti-CD3 (25 µg/ml final concentration) and anti-mouse CD28 (2 µg/ml final concentration) in the presence of BD GolgiStop™ (2 µM final concentration; Cat. No. 554724). The cells were then stained with 0.06 µg of FITC rat anti-mouse CD4 (FITC-RM4-5, Cat. No. 553047) fixed, permeabilized, and subsequently stained with 0.12 µg of PE rat anti-mouse GM-CSF antibody by using the Pharmingen staining protocol (left panel). To demonstrate specificity of staining, the binding of PE-MP1-22E9 was blocked by the preincubation of the conjugated antibody with recombinant mouse GM-CSF (1 µg, Cat. No. 554586; center panel), and by preincubation of the fixed/permeabilized cells with the unlabelled MP1-22E9 mouse antibody (10 µg, Cat. No. 556916; right panel). The quadrant markers for the bivariate dot plots were set based on the autofluorescence control, and verified with the recombinant cytokine blocking (center panel) and unlabelled antibody blocking (right panel) specificity controls.

Product Details
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BD Pharmingen™
Mouse (QC Testing)
Rat IgG2a
Recombinant Mouse GM-CSF
Flow cytometry (Routinely Tested), ELISA Capture (Tested During Development)
0.2 mg/ml
AB_395371
Aqueous buffered solution containing ≤0.09% sodium azide.
RUO


Preparation And Storage

Store undiluted at 4°C and protected from prolonged exposure to light. Do not freeze. 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.

Recommended Assay Procedures

Immunofluorescent Staining and Flow Cytometric Analysis: The MP1-22E9 antibody is useful for immunofluorescent staining and flow cytometric analysis to identify and enumerate GM-CSF producing cells within mixed cell populations. The PE-conjugated MP1-22E9 antibody (Cat. No. 554406) is especially suitable for these experiments (see Figure). For optimal immunofluorescent staining and flow cytometric analysis, this anti-cytokine antibody should be titrated (≤0.5 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 PE-MP1-22E9 antibody with ligand (e.g., mouse GM-CSF, Cat. No. 554586) prior to staining, or 2) pre-block paraformaldehyde-fixed/saponin-permeabilized cells with unlabeled MP1-22E9 antibody (Cat. No. 554404) prior to staining. The intracellular staining technique and use of blocking controls are described in detail by C. Prussin and D. Metcalfe. A suitable rat IgG2a isotype control immunoglobulin for assessing the level of background staining on paraformaldehyde-fixed/saponin-permeabilized mouse cells is the PE-R35-95 antibody (Cat. No. 554689); use at comparable concentrations to antibody of interest

ELISA Capture: The purified MP1-22E9 antibody (Cat. No. 554404) is useful as a capture antibody for a sandwich ELISA for measuring mouse GM-CSF protein levels. The purified MP1-22E9 antibody can be paired with the biotinylated MP1-31G6 antibody (Cat. No. 554407) as the detection antibody, with recombinant mouse GM-CSF (Cat. No. 554586) as the standard.

Neutralization: The NA/LE™ MP1-22E9 antibody (Cat. No. 554403) is useful for neutralization of mouse GM-CSF bioactivity. A suitable NA/LE™ rat IgG2a isotype control to match the MP1-22E9 antibody is the R35-95 antibody, Cat. No. 554687.

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.
  4. Use of these products to measure activation antigens expressed on mononuclear cell subsets for the purpose of monitoring immunoregulatory status can fall under one or more claims of the following patents: US Patent Nos. 5,445,939, 5,656,446, 5,843,689; European Patent No. 319,543; Canadian Patent No. 1,296,622; Australian Patent No. 615,880; and Japanese Patent No. 2,769,156.
  5. For fluorochrome spectra and suitable instrument settings, please refer to our Multicolor Flow Cytometry web page at www.bdbiosciences.com/colors.
  6. An isotype control should be used at the same concentration as the antibody of interest.
554406 Rev. 2
Antibody Details
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MP1-22E9

The MP1-22E9 monoclonal antibody specifically binds to mouse Granulocyte-Macrophage Colony Stimulating Factor (GM-CSF). The immunogen used to generate the MP1-22E9 hybridoma was yeast-expressed recombinant mouse GM-CSF. This is a neutralizing antibody.

554406 Rev. 2
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
554406 Rev.2
Citations & References
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Development References (4)

  1. Nozaki S, Abrams JS, Pearce MK, Sauder DN. Augmentation of granulocyte/macrophage colony-stimulating factor expression by ultraviolet irradiation is mediated by interleukin 1 in Pam 212 keratinocytes. J Invest Dermatol. 1991 July; 97(1):10-14. (Clone-specific: ELISA). View Reference
  2. 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
  3. 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
  4. Suda T, O'Garra A, MacNeil I, Fischer M, Bond MW, Zlotnik A. Identification of a novel thymocyte growth-promoting factor derived from B cell lymphomas. Cell Immunol. 1990; 129(1):228-240. (Clone-specific). View Reference
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554406 Rev. 2

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