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BB515 Mouse Anti-Human CD215 (IL-15Rα)
BB515 Mouse Anti-Human CD215 (IL-15Rα)

Multiparameter flow cytometric analysis of CD215 (IL15Rα) expression on human monocytes. Human peripheral blood mononuclear cells were cultured in complete tissue culture medium without (Left Plots) or with (Right Plots) lipopolysaccharide (LPS, 100 ng/ml) and Recombinant Human IFN-γ protein (Cat. No. 554617, 10 ng/ml) for 20 hours at 37°C. The cells were harvested, preincubated with Human BD Fc Block™ (Cat. No. 564219), and then stained with APC Mouse Anti-Human CD14 antibofy (Cat. No. 555399) and with either BD Horizon™ BB515 Mouse IgG2b, κ Isotype Control (Cat. No. 564510; Upper Plots) or BD Horizon™ BB515 Mouse Anti-Human CD215 (IL15Rα) antibody (Cat. No. 567746/567747; Lower Plots). BD Via-Probe™ Cell Viability 7-AAD Solution (Cat. No. 555815/555816) was added to cells right before analysis. Bivariate pseudocolor density plots showing the correlated expression of CD215 (IL15Rα) [or Ig Isotype control staining] versus CD14 were derived from gated events with the forward and side light-scatter characteristics of viable (7-AAD negative ) monocytes. Flow cytometry and data analysis were performed using a BD LSRFortessa™ X-20 Cell Analyzer System and FlowJo™ software.

Multiparameter flow cytometric analysis of CD215 (IL15Rα) expression on human monocytes. Human peripheral blood mononuclear cells were cultured in complete tissue culture medium without (Left Plots) or with (Right Plots) lipopolysaccharide (LPS, 100 ng/ml) and Recombinant Human IFN-γ protein (Cat. No. 554617, 10 ng/ml) for 20 hours at 37°C. The cells were harvested, preincubated with Human BD Fc Block™ (Cat. No. 564219), and then stained with APC Mouse Anti-Human CD14 antibofy (Cat. No. 555399) and with either BD Horizon™ BB515 Mouse IgG2b, κ Isotype Control (Cat. No. 564510; Upper Plots) or BD Horizon™ BB515 Mouse Anti-Human CD215 (IL15Rα) antibody (Cat. No. 567746/567747; Lower Plots). BD Via-Probe™ Cell Viability 7-AAD Solution (Cat. No. 555815/555816) was added to cells right before analysis. Bivariate pseudocolor density plots showing the correlated expression of CD215 (IL15Rα) [or Ig Isotype control staining] versus CD14 were derived from gated events with the forward and side light-scatter characteristics of viable (7-AAD negative ) monocytes. Flow cytometry and data analysis were performed using a BD LSRFortessa™ X-20 Cell Analyzer System and FlowJo™ software.

Product Details
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BD Horizon™
IL15RA; IL-15R-alpha; IL-15RA; IL-15Rα; CD215
Human (QC Testing)
Mouse BALB/c IgG2b, κ
Recombinant Human IL-15Rα Extracellular Domain
Flow cytometry (Routinely Tested)
5 µl
IX 234
3601
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 2 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. Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
  2. 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.
  3. 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).
  4. For fluorochrome spectra and suitable instrument settings, please refer to our Multicolor Flow Cytometry web page at www.bdbiosciences.com/colors.
  5. An isotype control should be used at the same concentration as the antibody of interest.
  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.
567746 Rev. 1
Antibody Details
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JM7A4

The JM7A4 monoclonal antibody specifically binds to CD215, which is also known as the IL-15 Receptor alpha subunit (IL-15R-alpha, IL-15Ra, or IL-15Rα). This type I transmembrane glycoprotein is encoded by IL15RA (Interleukin 15 receptor subunit alpha) and is variably expressed on macrophages, natural killer (NK) cells, T cells and B cells and by some nonlymphoid cells including fibroblasts.  Although it can independently bind IL-15 with high affinity, it does not contain a signaling motif. CD215 (IL-15Rα) can present IL-15 in cis or trans fashion to the IL-2/15R beta (CD122) and IL-2R gamma (γc or CD132) receptor complex which can then transduce signals intracellularly. Several different CD215 (IL-15Rα) isoforms have been described that are produced by alternative splicing and may alter signal transduction responses to IL-15. A cleaved soluble form of CD215 known as sIL-15RA has also been reported which can bind and antagonize IL-15 activity. By binding to its heterotrimeric receptor, IL-15 plays crucial roles in innate immunity, eg, through the activation of NK cells and adaptive immunity, eg, in enhancing the survival of CD8+ memory T cells.

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 FluorTM 488 cannot be used simultaneously. It is not recommended to use BB515 in cocktails that include Streptavidin conjugates as it may cause high background.

567746 Rev. 1
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
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Citations & References
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Development References (5)

  1. Dubois S, Mariner J, Waldmann TA, Tagaya Y. IL-15Ralpha recycles and presents IL-15 In trans to neighboring cells.. Immunity. 2002; 17(5):537-47. (Immunogen: Flow cytometry). View Reference
  2. Llinas L, Lazaro A, de Salort J, Matesanz-Isabel J, Sintes J, Engel P. Expression profiles of novel cell surface molecules on B-cell subsets and plasma cells as analyzed by flow cytometry. Immunol Lett. 2011; 134(2):113-121. (Clone-specific: Flow cytometry, Immunohistochemistry). View Reference
  3. Matesanz-Isabel J, Sintes J, Llinas L, de Salort J, Lazaro A, Engel P. New B-cell CD molecules. Immunol Lett. 2011; 134(2):104-112. (Clone-specific: Flow cytometry). View Reference
  4. Mortier E, Bernard J, Plet A, Jacques Y. Natural, proteolytic release of a soluble form of human IL-15 receptor alpha-chain that behaves as a specific, high affinity IL-15 antagonist.. J Immunol. 2004; 173(3):1681-8. (Biology). View Reference
  5. Vámosi G, Bodnár A, Vereb G, et al. IL-2 and IL-15 receptor alpha-subunits are coexpressed in a supramolecular receptor cluster in lipid rafts of T cells.. Proc Natl Acad Sci USA. 2004; 101(30):11082-7. (Clone-specific: Immunofluorescence). View Reference
View All (5) View Less
567746 Rev. 1

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