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BB515 Mouse Anti-Human CD152
BB515 Mouse Anti-Human CD152
Multiparameter flow cytometric analysis of CD152 expression on activated Human peripheral blood lymphocytes. Phytohemagglutinin (PHA)-activated (3 days; Stimulated) human peripheral blood mononuclear cells were preincubated with BD Pharmingen™ Human BD Fc Block™ (Cat. No. 564219/564220). These cells were then stained with APC Mouse Anti-Human CD3 antibody (Cat. No. 555335/561810/561811) and with either BD Horizon™ BB515 Mouse IgG2a, κ Isotype Control (Cat. No. 564515; Left Plot) or BD Horizon™ BB515 Mouse Anti-Human CD152 antibody (Cat. No. 566917/566918; Right Plot). BD Via-Probe™ Cell Viability 7-AAD Solution (Cat. No. 555815/555816) was added to cells right before analysis. The bivariate pseudocolor density plot showing the correlated expression of CD152 (or Ig Isotype control staining) versus CD3 was derived from gated events with the forward and side light-scatter characteristics of viable (DAPI-negative) lymphocytes. Flow cytometry and data analysis were performed using a BD LSRFortessa™ Cell Analyzer System and FlowJo™ software.
Multiparameter flow cytometric analysis of CD152 expression on activated Human peripheral blood lymphocytes. Phytohemagglutinin (PHA)-activated (3 days; Stimulated) human peripheral blood mononuclear cells were preincubated with BD Pharmingen™ Human BD Fc Block™ (Cat. No. 564219/564220). These cells were then stained with APC Mouse Anti-Human CD3 antibody (Cat. No. 555335/561810/561811) and with either BD Horizon™ BB515 Mouse IgG2a, κ Isotype Control (Cat. No. 564515; Left Plot) or BD Horizon™ BB515 Mouse Anti-Human CD152 antibody (Cat. No. 566917/566918; Right Plot). BD Via-Probe™ Cell Viability 7-AAD Solution (Cat. No. 555815/555816) was added to cells right before analysis. The bivariate pseudocolor density plot showing the correlated expression of CD152 (or Ig Isotype control staining) versus CD3 was derived from gated events with the forward and side light-scatter characteristics of viable (DAPI-negative) lymphocytes. Flow cytometry and data analysis were performed using a BD LSRFortessa™ Cell Analyzer System and FlowJo™ software.
Product Details
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BD Horizon™
CTLA-4; AILIM; Cytotoxic T-lymphocyte protein 4
Human (QC Testing), Rhesus,Cynomolgus,Baboon (Tested in Development)
Mouse BALB/c IgG2a, κ
Human CTLA4 Recombinant Protein
Flow cytometry (Routinely Tested), Intracellular staining (flow cytometry) (Tested During Development)
5 µl
IX 34
1493
AB_2869947
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 BD® 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 BD® CompBeads 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. 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).
  3. An isotype control should be used at the same concentration as the antibody of interest.
  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. For fluorochrome spectra and suitable instrument settings, please refer to our Multicolor Flow Cytometry web page at www.bdbiosciences.com/colors.
  6. Species cross-reactivity detected in product development may not have been confirmed on every format and/or application.
  7. 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.
  8. Please refer to http://regdocs.bd.com to access safety data sheets (SDS).
  9. Alexa Fluor™ is a trademark of Life Technologies Corporation.
  10. For U.S. patents that may apply, see bd.com/patents.
566918 Rev. 3
Antibody Details
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BNI3

The BNI3 monoclonal antibody specifically binds to the human cytolytic T lymphocyte-associated antigen (CTLA-4), also known as CD152. CTLA-4 is transiently expressed on activated CD28+ T cells and binds to CD80 and CD86 present on antigen presenting cells (APC) with high avidity. This interaction appears to deliver a negative regulatory signal to the T cell. Recent reports indicate that CTLA-4 is also expressed on B cells when cultured with activated T cells, suggesting a role for CTLA-4 in the regulation of B-cell response. Immobilized BNI3 antibody enhances T-cell proliferation induced by antibody-mediated crosslinking of CD3 and CD28. Recent studies have shown that CD152 can be expressed by regulatory T (Treg) cells. After cellular fixation and permeabilization, the BNI3 antibody can stain intracellular CD152 expressed in T cells including Treg cells. Clone BNI3 was studied in the VI Leukocyte Typing Workshop.

566918 Rev. 3
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.
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BB515
Blue 488 nm
490 nm
515 nm
566918 Rev.3
Citations & References
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Development References (10)

  1. Cabezon R, Sintes J, Llinas L, Benitez-Ribas D. Analysis of HLDA9 mAbs on plasmacytoid dendritic cell. Immunol Lett. 2011; 134(2):167-173. (Clone-specific: Flow cytometry). View Reference
  2. Castan J, Klauenberg U, Kalmar P, Fleischer B, Broker BM. Expression of CTLA-4 (CD152) on human medullary CD4+ thymocytes. Med Microbiol Immunol (Berl). 1998; 187(1):49-52. (Immunogen: Fluorescence microscopy, Immunocytochemistry, Immunofluorescence, Immunohistochemistry). View Reference
  3. Castan J, Tenner-Racz K, Racz P, Fleischer B, Broker BM. Accumulation of CTLA-4 expressing T lymphocytes in the germinal centres of human lymphoid tissues. Immunology. 1997; 90(2):265-271. (Immunogen: ELISA, Fluorescence microscopy, Immunofluorescence, Immunohistochemistry). View Reference
  4. Healy ZR, Murdoch DM. OMIP-036: Co-inhibitory receptor (immune checkpoint) expression analysis in human T cell subsets.. Cytometry A. 2016; 89(10):889-892. (Clone-specific: Intracellular Staining/Flow Cytometry). View Reference
  5. Kuiper HM, Brouwer M, Linsley PS, van Lier RA. Activated T cells can induce high levels of CTLA-4 expression on B cells. J Immunol. 1995; 155(4):1776-1783. (Biology). View Reference
  6. Lindsten T, Lee KP, Harris ES, et al. Characterization of CTLA-4 structure and expression on human T cells. J Immunol. 1993; 151(7):3489-3499. (Biology). View Reference
  7. Morton PA, Fu XT, Stewart JA, et al. Differential effects of CTLA-4 substitutions on the binding of human CD80 (B7-1) and CD86 (B7-2). J Immunol. 1996; 156(3):1047-1054. (Biology). View Reference
  8. Rabe H, Lundell AC, Andersson K, Adlerberth I, Wold AE, Rudin A. Higher proportions of circulating FOXP3+ and CTLA-4+ regulatory T cells are associated with lower fractions of memory CD4+ T cells in infants.. J Leukoc Biol. 2011; 90(6):1133-40. (Clone-specific: Intracellular Staining/Flow Cytometry). View Reference
  9. Santegoets SJ, Dijkgraaf EM, Battaglia A, et al. Monitoring regulatory T cells in clinical samples: consensus on an essential marker set and gating strategy for regulatory T cell analysis by flow cytometry.. Cancer Immunol Immunother. 2015; 64(10):1271-86. (Clone-specific: Intracellular Staining/Flow Cytometry). View Reference
  10. Wang H, Shih CC, Waters JB, et al. CD152 (CTLA4) Workshop: Expression and function of CD152 on human T cells: A study using a mouse anti-human CD152 monoclonal antibody BNI3.1. 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.; 1997:97-98.
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566918 Rev. 3

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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.