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BB515 Mouse Anti-Human IL-3Rα (CD123)
BB515 Mouse Anti-Human IL-3Rα (CD123)

Multiparameter flow cytometric analysis of IL-3Rα (CD123) expression on human peripheral blood leucocyte populations. Human whole blood was stained with APC Mouse Anti-Human CD45RO (Cat No. 559865/560899) and with either BD Horizon™ BB515 Mouse IgG1, κ Isotype Control (Cat No. 564416; Left Plots) or BD Horizon™ BB515 Mouse Anti-Human IL-3Rα (CD123) (Cat No. 567715/567716; Right Plots). Erythrocytes were lysed with BD FACS™ Lysing Solution (Cat No. 349202). Flow cytometry and data analysis were performed using a BD LSRFortessa™ Cell Analyzer System and FlowJo™ software.

Upper Plots: Bivariate pseudocolor density plots showing the correlated expression of IL-3Rα (CD123) [or Ig Isotype control staining] versus side light-scatter (SSC-A) signals were derived from gated events with the forward and side light-scatter characteristics of intact human leucocytes.

Lower Plots: Bivariate pseudocolor density plots showing the correlated expression of IL-3Rα (CD123) [or Ig Isotype control staining] versus CD45RO were derived from gated events with the forward and side light-scatter characteristics of intact human lymphocytes.

Multiparameter flow cytometric analysis of IL-3Rα (CD123) expression on human peripheral blood leucocyte populations. Human whole blood was stained with APC Mouse Anti-Human CD45RO (Cat No. 559865/560899) and with either BD Horizon™ BB515 Mouse IgG1, κ Isotype Control (Cat No. 564416; Left Plots) or BD Horizon™ BB515 Mouse Anti-Human IL-3Rα (CD123) (Cat No. 567715/567716; Right Plots). Erythrocytes were lysed with BD FACS™ Lysing Solution (Cat No. 349202). Flow cytometry and data analysis were performed using a BD LSRFortessa™ Cell Analyzer System and FlowJo™ software.

Upper Plots: Bivariate pseudocolor density plots showing the correlated expression of IL-3Rα (CD123) [or Ig Isotype control staining] versus side light-scatter (SSC-A) signals were derived from gated events with the forward and side light-scatter characteristics of intact human leucocytes.

Lower Plots: Bivariate pseudocolor density plots showing the correlated expression of IL-3Rα (CD123) [or Ig Isotype control staining] versus CD45RO were derived from gated events with the forward and side light-scatter characteristics of intact human lymphocytes.

Product Details
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BD Horizon™
IL3RA; IL3R; IL-3R-alpha; IL-3RA
Human (QC Testing)
Mouse BALB/c IgG1, κ
Human IL3RA Transfected Cell Line
Flow cytometry (Routinely Tested)
5 µl
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 to the dye under optimum conditions and unconjugated antibody and free dye were removed.

Recommended Assay Procedures

BD® CompBeads can be used as surrogates to assess fluorescence spillover (Compensation). When fluorochrome conjugated antibodies are bound to CompBeads, they have spectral properties very similar to cells.  However, for some fluorochromes there can be small differences in spectral emissions compared to cells, resulting in spillover values that differ when compared to biological controls. It is strongly recommended that when using a reagent for the first time, users compare the spillover on cells and CompBead to ensure that BD® CompBeads are appropriate for your specific cellular application.

For optimal and reproducible results, BD Horizon Brilliant™ Stain Buffer should be used anytime BD Horizon Brilliant™ dyes are used in a multicolor flow cytometry panel. Fluorescent dye interactions may cause staining artifacts which may affect data interpretation. The BD Horizon Brilliant Stain Buffer was designed to minimize these interactions. When BD Horizon Brilliant Stain Buffer is used in in the multicolor panel, it should also be used in the corresponding compensation controls for all dyes to achieve the most accurate compensation. For the most accurate compensation, compensation controls created with either cells or beads should be exposed to BD Horizon Brilliant Stain Buffer for the same length of time as the corresponding multicolor panel. More information can be found in the Technical Data Sheet of the BD Horizon Brilliant Stain Buffer (Cat. No. 563794/566349) or the BD Horizon Brilliant Stain Buffer Plus (Cat. No. 566385).

For optimal results, it is recommended to perform two washes after staining with antibodies. Cells may be prepared, stained with antibodies and washed twice with wash buffer per established protocols for immunofluorescence staining, prior to acquisition on a flow cytometer. Performing fewer than the recommended wash steps may lead to increased spread of the negative population.

Product Notices

  1. This reagent has been pre-diluted for use at the recommended Volume per Test. We typically use 1 × 10^6 cells in a 100-µl experimental sample (a test).
  2. An isotype control should be used at the same concentration as the antibody of interest.
  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. Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
  5. For fluorochrome spectra and suitable instrument settings, please refer to our Multicolor Flow Cytometry web page at www.bdbiosciences.com/colors.
  6. BD Horizon Brilliant Stain Buffer is covered by one or more of the following US patents: 8,110,673; 8,158,444; 8,575,303; 8,354,239.
  7. Please refer to http://regdocs.bd.com to access safety data sheets (SDS).
  8. Alexa Fluor™ is a trademark of Life Technologies Corporation.
Antibody Details
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6H6

The 6H6 monoclonal antibody specifically recognizes the Interleukin-3 receptor alpha chain (IL-3Ra) which is also known as CD123. IL-3Ra (CD123) is a ~70 kDa type I transmembrane glycoprotein that is encoded by IL3RA (interleukin 3 receptor subunit alpha) which belongs to the type I cytokine receptor family within the immunoglobulin gene superfamily. This receptor chain consists of an extracellular region that contains an immunoglobulin-like N-terminal domain (NTD) with a fibronectin type III (FnIII) fold followed by two more FnIII domains that form the cytokine receptor module (CRM), a transmembrane region, and an intracellular tail. IL-3Ra (CD123) binds IL-3 specifically and with low affinity. IL-3Ra (CD123) forms a high-affinity signaling receptor for IL-3 (IL-3R) with the ß common chain (ßc; also known as, CD131) that is shared with the heterodimeric IL-5 and granulocyte-macrophage colony-stimulating factor (GM-CSF) receptors. IL-3Ra (CD123) is variably expressed on certain hematopoietic progenitor cells, basophils, eosinophils, mast cells, monocytes, macrophages, dendritic cells, megakaryocytes, and on some B cells, endothelial cells, and leukemia cells. Bound IL-3 can signal through IL-3R to promote the activation, proliferation, differentiation, and viability of these cells. Amongst monoclonal antibodies specific for human IL-3Ra (CD123), the 6H6 and 9F5 antibodies do not block IL-3 binding to the IL-3R whereas the 7G3 antibody does block IL-3 binding to its receptor in a dose-dependent manner.

The antibody was conjugated to BD Horizon™ BB515 which is part of the BD Horizon Brilliant™ Blue family of dyes. With an Ex Max near 490 nm and an Em Max near 515 nm, BD Horizon™ BB515 can be excited by the blue laser (488 nm) laser and detected with a 530/30 nm filter. This dye has been exclusively developed by BD Biosciences and is up to seven times brighter than FITC with less spillover into the PE channel. Due to similar excitation and emission properties, BB515, FITC, and Alexa Fluor™ 488 cannot be used simultaneously. It is not recommended to use BB515 in cocktails that include Streptavidin conjugates as it may cause high background.

Format Details
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BB515
The BD Horizon Brilliant™ Blue 515 (BB515) dye is part of the BD Horizon Brilliant™ Blue family of dyes. This dye is a polymer fluorochrome with an excitation maximum (Ex Max) at 490-nm and an emission maximum (Em Max) of 515-nm. Driven by BD innovation, BB515 is designed to be excited by the blue laser (488-nm) and detected using an optical filter centered near 520-nm (e.g., 530/30-nm). BB515 reagents are significantly brighter than equivalent FITC or Alexa Fluor™ 488 reagents with less spillover into the PE detector. Please ensure that your instrument’s configurations (lasers and optical filters) are appropriate for this dye.
altImg
BB515
Blue 488 nm
490 nm
515 nm
Citations & References
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Development References (7)

  1. Broughton SE, Hercus TR, Hardy MP, et al. Dual mechanism of interleukin-3 receptor blockade by an anti-cancer antibody.. Cell Rep. 2014; 8(2):410-9. (Biology). View Reference
  2. Kohrgruber N, Halanek N, Gröger M, et al. Survival, maturation, and function of CD11c- and CD11c+ peripheral blood dendritic cells are differentially regulated by cytokines.. J Immunol. 1999; 163(6):3250-9. (Biology). View Reference
  3. Macardle PJ, Chen Z, Shih CY, et al. Characterization of human leucocytes bearing the IL-3 receptor. Cell Immunol. 1996; 168(1):59-68. (Biology). View Reference
  4. Miyajima A. CDw123 (Interleukin 3 receptor α chain) Workshop Panel report. In: Kishimoto T. Tadamitsu Kishimoto .. et al., ed. Leucocyte typing VI : white cell differentiation antigens : proceedings of the sixth international workshop and conference held in Kobe, Japan, 10-14 November 1996. New York: Garland Pub.; 1997:854-855.
  5. Sun Q, Woodcock JM, Rapoport A, et al. Monoclonal antibody 7G3 recognizes the N-terminal domain of the human interleukin-3 (IL-3) receptor alpha-chain and functions as a specific IL-3 receptor antagonist.. Blood. 1996; 87(1):83-92. (Immunogen: Flow cytometry, Immunoprecipitation, Western blot). View Reference
  6. Yamada T, Sun Q, Zeibecoglou K, et al. IL-3, IL-5, granulocyte-macrophage colony-stimulating factor receptor alpha-subunit, and common beta-subunit expression by peripheral leukocytes and blood dendritic cells.. J Allergy Clin Immunol. 1998; 101(5):677-82. (Clone-specific: Flow cytometry). View Reference
  7. Zola H. Leukocyte and stromal cell molecules : the CD markers. Hoboken, N.J.: Wiley-Liss; 2007.
View All (7) View Less

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For Research Use Only. Not for use in diagnostic or therapeutic procedures.