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BV605 Armenian Hamster Anti-ICOS (CD278)
BV605 Armenian Hamster Anti-ICOS (CD278)
Multicolor Flow Cytometric Analysis of ICOS (CD278) Expression    Left Panel - ICOS (CD278) expression on human peripheral blood lymphocytes (PBL). Whole blood was stained with APC Mouse Anti-Human CD3 antibody (Cat. No. 555335) and either no antibody (BD Horizon™ BV605 Autofluorescence Control; Top Plot) or BD Horizon™ BV605 Hamster Anti-ICOS (CD278) antibody (Cat. No. 567920/567921; Bottom Plot) at 0.25 µg/test. Erythrocytes were lysed with BD FACS Lysing Solution (Cat. No. 349202). The pseudocolor density plot showing the correlated expression of ICOS (CD278) [or BD Horizon™ BV605 Autofluorescence] versus CD3 was derived from gated events with the forward and side light-scatter characteristics of intact lymphocytes.    Right Panel - ICOS (CD278) expression on mouse thymocyte subsets. Mouse thymocytes were preincubated with Purified Rat Anti-Mouse CD16/CD32 antibody (Mouse BD Fc Block™) (Cat. No. 553142). The cells were then stained with FITC Rat anti-Mouse CD4 (Cat. No. 553729) and APC Rat Anti-Mouse CD8a (Cat. No. 553035) antibodies, and either no antibody (BD Horizon™ BV605 Autofluorescence Control; dashed line histograms) or BD Horizon BV605 Hamster Anti-ICOS (CD278) antibody (solid line histograms) at 0.25 µg/test. The fluorescence histograms showing ICOS (CD278) expression (or BD Horizon™ BV605 Autofluorescence) were derived from CD4 and CD8 gated events with the forward and side light-scatter characteristics of viable thymocytes as indicated.    Flow cytometry and data analysis were performed using a BD LSRFortessa™ Cell Analyzer System and FlowJo™ software. Data shown on this Technical Data Sheet are not lot specific.
Multicolor Flow Cytometric Analysis of ICOS (CD278) Expression    Left Panel - ICOS (CD278) expression on human peripheral blood lymphocytes (PBL). Whole blood was stained with APC Mouse Anti-Human CD3 antibody (Cat. No. 555335) and either no antibody (BD Horizon™ BV605 Autofluorescence Control; Top Plot) or BD Horizon™ BV605 Hamster Anti-ICOS (CD278) antibody (Cat. No. 567920/567921; Bottom Plot) at 0.25 µg/test. Erythrocytes were lysed with BD FACS Lysing Solution (Cat. No. 349202). The pseudocolor density plot showing the correlated expression of ICOS (CD278) [or BD Horizon™ BV605 Autofluorescence] versus CD3 was derived from gated events with the forward and side light-scatter characteristics of intact lymphocytes.    Right Panel - ICOS (CD278) expression on mouse thymocyte subsets. Mouse thymocytes were preincubated with Purified Rat Anti-Mouse CD16/CD32 antibody (Mouse BD Fc Block™) (Cat. No. 553142). The cells were then stained with FITC Rat anti-Mouse CD4 (Cat. No. 553729) and APC Rat Anti-Mouse CD8a (Cat. No. 553035) antibodies, and either no antibody (BD Horizon™ BV605 Autofluorescence Control; dashed line histograms) or BD Horizon BV605 Hamster Anti-ICOS (CD278) antibody (solid line histograms) at 0.25 µg/test. The fluorescence histograms showing ICOS (CD278) expression (or BD Horizon™ BV605 Autofluorescence) were derived from CD4 and CD8 gated events with the forward and side light-scatter characteristics of viable thymocytes as indicated.    Flow cytometry and data analysis were performed using a BD LSRFortessa™ Cell Analyzer System and FlowJo™ software. Data shown on this Technical Data Sheet are not lot specific.
Product Details
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BD Horizon™
ICOS; AILIM; H4; Inducible T-cell costimulator; CVID1; Ly115
Human (QC Testing), Mouse (Tested in Development), Rat, Rhesus (Reported)
Armenian Hamster IgG
Mouse D10.G4.1 T-cell clone
Flow cytometry (Routinely Tested)
0.2 mg/ml
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 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).

Product Notices

  1. Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
  2. Please observe the following precautions: Absorption of visible light can significantly alter the energy transfer occurring in any tandem fluorochrome conjugate; therefore, we recommend that special precautions be taken (such as wrapping vials, tubes, or racks in aluminum foil) to prevent exposure of conjugated reagents, including cells stained with those reagents, to room illumination.
  3. Source of all serum proteins is from USDA inspected abattoirs located in the United States.
  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.
  5. Since applications vary, each investigator should titrate the reagent to obtain optimal results.
  6. For fluorochrome spectra and suitable instrument settings, please refer to our Multicolor Flow Cytometry web page at www.bdbiosciences.com/colors.
  7. An isotype control should be used at the same concentration as the antibody of interest.
  8. CF™ is a trademark of Biotium, Inc.
  9. Although every effort is made to minimize the lot-to-lot variation in the efficiency of the fluorochrome energy transfer, differences in the residual emission from BD Horizon™ BV421 may be observed. Therefore, we recommend that individual compensation controls be performed for every BD Horizon™ BV605 conjugate.
  10. Species cross-reactivity detected in product development may not have been confirmed on every format and/or application.
  11. BD Horizon Brilliant Violet 605 is covered by one or more of the following US patents: 8,110,673; 8,158,444; 8,227,187; 8,455,613; 8,575,303; 8,354,239.
  12. 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.
  13. Please refer to http://regdocs.bd.com to access safety data sheets (SDS).
Antibody Details
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C398.4A

The C398.4A monoclonal antibody specifically binds to Inducible Costimulator (ICOS), which is also known as, CD278, Activation-inducible lymphocyte immunomediatory molecule (AILIM), or H4. ICOS is a type I transmembrane glycoprotein that forms a disulfide-linked homodimer and belongs to the CD28 family within the Ig superfamily. ICOS is expressed on either CD4+ or CD8+ single-positive mature thymocytes, T cells, or subsets of Innate Lymphoid Cells (ILC). Its expression is upregulated on activated T lymphocytes. ICOS is a costimulatory receptor that can bind to the B7-H2 ligand (CD275, ICOS-L) that is expressed on B cells, monocytes, macrophages, dendritic cells, and endothelial cells. ICOS plays a critical role in many types of T cell-dependent immunity. In the case of humoral immunity, for example, ICOS signaling is critical for the differentiation of T follicular helper (Tfh) cells and development of germinal centers. Although C398.4A was generated against mouse ICOS, this antibody reportedly crossreacts with human, rhesus, and rat ICOS.

Format Details
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BV605
The BD Horizon Brilliant Violet™ 605 (BV605) dye is part of the BD Horizon Brilliant Violet™ family of dyes. This tandem fluorochrome is comprised of a BV421 donor with an excitation maximum (Ex Max) of 407-nm and an acceptor dye with an emission maximum (Em Max) at 605-nm. BV605, driven by BD innovation, is designed to be excited by the violet laser (405-nm) and detected using an optical filter centered near 610-nm (e.g., a 610/20-nm bandpass filter). The acceptor dye can be excited by the yellow-green (561-nm) laser resulting in cross-laser excitation and fluorescence spillover. Please ensure that your instrument’s configurations (lasers and optical filters) are appropriate for this dye.
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BV605
Violet 405 nm
407 nm
605 nm
Citations & References
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Development References (11)

  1. Araujo LM, Fert I, Jouhault Q, et al. Increased production of interleukin-17 over interleukin-10 by treg cells implicates inducible costimulator molecule in experimental spondyloarthritis. Arthritis Rheum. 2014; 66(9):2412-2422. (Clone-specific: Flow cytometry). View Reference
  2. Brenchley JM, Vinton C, Tabb B, et al. Differential infection patterns of CD4+ T cells and lymphoid tissue viral burden distinguish progressive and nonprogressive lentiviral infections.. Blood. 2012; 120(20):4172-81. (Clone-specific: Flow cytometry). View Reference
  3. Buonfiglio D, Bragardo M, Bonissoni S, et al. Characterization of a novel human surface molecule selectively expressed by mature thymocytes, activated T cells and subsets of T cell lymphomas. Eur J Immunol. 1999; 29(9):2863-2879. (Clone-specific: Flow cytometry, Immunohistochemistry, Immunoprecipitation, Radioimmunoassay). View Reference
  4. Buonfiglio D, Bragardo M, Redoglia V, et al. The T cell activation molecule H4 and the CD28-like molecule ICOS are identical. Eur J Immunol. 2000; 30(12):3463-3467. (Clone-specific: Blocking, Flow cytometry). View Reference
  5. Chen Y, Shen S, Gorentla BK, Gao J, Zhong XP. Murine regulatory T cells contain hyperproliferative and death-prone subsets with differential ICOS expression. J Immunol. 2012; 188(4):1698-1707. (Clone-specific: Flow cytometry, Fluorescence activated cell sorting). View Reference
  6. Hutloff A, Dittrich AM, Beier KC, et al. ICOS is an inducible T-cell co-stimulator structurally and functionally related to CD28. Nature. 1999; 397(3716):263-266. (Biology). View Reference
  7. Klatt NR, Vinton CL, Lynch RM et al. SIV infection of rhesus macaques results in dysfunctional T- and B-cell responses to neo and recall Leishmania major vaccination. Blood. 2011; 118(22):5803-5812. (Clone-specific: Flow cytometry). View Reference
  8. McAdam AJ, Chang TT, Lumelsky AE, et al. Mouse inducible costimulatory molecule (ICOS) expression is enhanced by CD28 costimulation and regulates differentiation of CD4+ T cells.. J Immunol. 2000; 165(9):5035-40. (Biology). View Reference
  9. Redoglia V, Dianzani U, Rojo JM, et al. Characterization of H4: a mouse T lymphocyte activation molecule functionally associated with the CD3/T cell receptor. Eur J Immunol. 1996; 26(11):2781-2789. (Immunogen: Bioassay, (Co)-stimulation, Flow cytometry, Functional assay, Immunoprecipitation, Radioimmunoassay). View Reference
  10. Sonnenberg GF, Mjosberg J, Spits H, Artis D. SnapShot: innate lymphoid cells. Immunity. 2013; 39(3):622-623. (Biology). View Reference
  11. Xu H, Wang X, Lackner AA, Veazey RS. PD-1(HIGH) Follicular CD4 T Helper Cell Subsets Residing in Lymph Node Germinal Centers Correlate with B Cell Maturation and IgG Production in Rhesus Macaques. Front Biosci. 2014; 5(85):1-7. (Clone-specific: Flow cytometry). View Reference
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For Research Use Only. Not for use in diagnostic or therapeutic procedures.