Two-color flow cytometric analysis of GM-CSF expression in stimulated mouse T cells. Mouse splenic CD4+ T cells were cultured (2 days) with plate-bound Purified NA/LE Hamster Anti-Mouse CD3e (Cat. No. 553057; 10 μg/ml for coating) and soluble Purified NA/LE Hamster Anti-Mouse CD28 (Cat. No. 553294; 2 μg/ml) antibodies with Recombinant Mouse IL-2 (Cat. No. 550069; 10 ng/ml) and Mouse IL-4 (Cat. No. 550067; 25 ng/ml). The cells were subsequently cultured (3 days) with Recombinant IL-2 and IL-4. The cells were then stimulated with PMA (Sigma Cat. No. P-8139) and Ionomycin (Sigma Cat. No. I-0634) in the presence of BD GolgiStop™ Protein Transport Inhibitor (containing Monensin) (Cat. No. 554724) for 6 hours. The cells were harvested, washed, and fixed with BD Cytofix™ Fixation Buffer (Cat. No. 554655). The cells were then washed and stained in BD Perm/Wash™ Buffer (Cat. No. 554723) with APC Rat Anti-Mouse CD4 antibody (Cat. No. 553051/561091) and either BD Horizon™ BV421 Rat IgG2a, κ Isotype Control (Cat. No. 562602; Left Panel) or BD Horizon BV421 Rat Anti-Mouse GM-CSF antibody (Cat. No. 564747; Right Panel). Two-color flow cytometric dot plots showing correlated expression of GM-CSF (or Ig Isotype control staining) versus CD4 were derived from gated events with the forward and side light-scatter characteristics of intact stimulated lymphocytes. Flow cytometric analysis was performed using a BD™ LSR II Flow Cytometer System.
Two-color flow cytometric analysis of GM-CSF expression in stimulated mouse T cells. Mouse splenic CD4+ T cells were cultured (2 days) with plate-bound Purified NA/LE Hamster Anti-Mouse CD3e (Cat. No. 553057; 10 μg/ml for coating) and soluble Purified NA/LE Hamster Anti-Mouse CD28 (Cat. No. 553294; 2 μg/ml) antibodies with Recombinant Mouse IL-2 (Cat. No. 550069; 10 ng/ml) and Mouse IL-4 (Cat. No. 550067; 25 ng/ml). The cells were subsequently cultured (3 days) with Recombinant IL-2 and IL-4. The cells were then stimulated with PMA (Sigma Cat. No. P-8139) and Ionomycin (Sigma Cat. No. I-0634) in the presence of BD GolgiStop™ Protein Transport Inhibitor (containing Monensin) (Cat. No. 554724) for 6 hours. The cells were harvested, washed, and fixed with BD Cytofix™ Fixation Buffer (Cat. No. 554655). The cells were then washed and stained in BD Perm/Wash™ Buffer (Cat. No. 554723) with APC Rat Anti-Mouse CD4 antibody (Cat. No. 553051/561091) and either BD Horizon™ BV421 Rat IgG2a, κ Isotype Control (Cat. No. 562602; Left Panel) or BD Horizon BV421 Rat Anti-Mouse GM-CSF antibody (Cat. No. 564747; Right Panel). Two-color flow cytometric dot plots showing correlated expression of GM-CSF (or Ig Isotype control staining) versus CD4 were derived from gated events with the forward and side light-scatter characteristics of intact stimulated lymphocytes. Flow cytometric analysis was performed using a BD™ LSR II Flow Cytometer System.
Product Details
BD Horizon™
Csf2; Csfgm; Colony stimulating factor 2 (granulocyte-macrophage)
Mouse (QC Testing)
Rat IgG2a
Recombinant Mouse GM-CSF
Intracellular staining (flow cytometry) (Routinely Tested)
0.2 mg/ml
AB_2738929
Aqueous buffered solution containing BSA and ≤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 BD Horizon BV421 under optimum conditions, and unconjugated antibody and free BD Horizon BV421 were removed.
Product Notices
- Since applications vary, each investigator should titrate the reagent to obtain optimal results.
- An isotype control should be used at the same concentration as the antibody of interest.
- 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.
- Source of all serum proteins is from USDA inspected abattoirs located in the United States.
- Pacific Blue™ is a trademark of Molecular Probes, Inc., Eugene, OR.
- For fluorochrome spectra and suitable instrument settings, please refer to our Multicolor Flow Cytometry web page at www.bdbiosciences.com/colors.
- Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
Companion Products
Stain Buffer (FBS) RUO
Size
500 mL
Cat No.
554656
Stain Buffer (BSA) RUO
Size
500 mL
Cat No.
554657
Fixation Buffer RUO
Size
100 mL
Cat No.
554655
Perm/Wash Buffer RUO
Size
100 mL
Cat No.
554723
Protein Transport Inhibitor (Containing Monensin) RUO
Size
0.7 mL
Cat No.
554724
BV421 Rat IgG2a, κ Isotype Control RUO
Size
50 µg
Cat No.
562602
564747 Rev. 1
Antibody Details
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.
The antibody was conjugated to BD Horizon BV421 which is part of the BD Horizon Brilliant™ Violet family of dyes. With an Ex Max of 407-nm and Em Max at 421-nm, BD Horizon BV421 can be excited by the violet laser and detected in the standard Pacific Blue™ filter set (eg, 450/50-nm filter). BD Horizon BV421 conjugates are very bright, often exhibiting a 10 fold improvement in brightness compared to Pacific Blue conjugates.
564747 Rev. 1
Format Details
BV421
The BD Horizon Brilliant Violet™ 421 (BV421) Dye is part of the BD Horizon Brilliant Violet™ family of dyes. This polymer-technology based dye has an excitation maximum (Ex Max) of 407-nm and an emission maximum (Em Max) at 423-nm. Driven by BD innovation, BV421 is designed to be excited by the violet laser (405-nm) and detected using an optical filter centered near 420-nm (e.g., a 431/28-nm or 450/50-nm bandpass filter). BV421 is an ideal alternative for V450 as it is approximately ten times brighter with less spillover into the BV510/V500 detector. Please ensure that your instrument’s configurations (lasers and optical filters) are appropriate for this dye.
BV421
Violet 405 nm
407 nm
423 nm
564747 Rev.1
Citations & References
Development References (4)
-
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
-
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: Flow cytometry). View Reference
-
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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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: Neutralization). View Reference
564747 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.
