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RB780 Mouse Anti-Human CD152
RB780 Mouse Anti-Human CD152
Multicolor flow cytometric analysis of CD152 expression on activated Human peripheral blood lymphocytes. Concanavalin A (Con A)-activated (3 days) Human peripheral blood mononuclear cells (PBMC) were preincubated with Human BD Fc Block™ (Cat. No. 564219/564220). These cells were then stained with APC Mouse Anti-Human CD3 antibody (Cat. No. 561811/561810) and with either BD Horizon™ RB780 Mouse IgG2a, κ Isotype Control (Cat. No. 568740; Left Plot) or BD Horizon™ RB780 Mouse Anti-Human CD152 antibody (Cat. No. 569335/569336; Right Plot). DAPI (4',6-Diamidino-2-Phenylindole, Dihydrochloride) Solution (Cat. No. 564907) 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™ X-20 Cell Analyzer System and FlowJo™ software.
Multicolor flow cytometric analysis of CD152 expression on activated Human peripheral blood lymphocytes. Concanavalin A (Con A)-activated (3 days) Human peripheral blood mononuclear cells (PBMC) were preincubated with Human BD Fc Block™ (Cat. No. 564219/564220). These cells were then stained with APC Mouse Anti-Human CD3 antibody (Cat. No. 561811/561810) and with either BD Horizon™ RB780 Mouse IgG2a, κ Isotype Control (Cat. No. 568740; Left Plot) or BD Horizon™ RB780 Mouse Anti-Human CD152 antibody (Cat. No. 569335/569336; Right Plot). DAPI (4',6-Diamidino-2-Phenylindole, Dihydrochloride) Solution (Cat. No. 564907) 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™ X-20 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/test
IX 34
1493
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 unreacted dye was 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.

Product Notices

  1. When using high concentrations of antibody, background binding of this dye to erythroid fragments produced by ammonium chloride-based lysis, such as with BD Pharm Lyse™ Lysing Buffer (Cat. No. 555899), has been observed when the antibody conjugate was present during the lysis procedure. This may cause nonspecific staining of target cells, such as leukocytes, which have bound the resulting erythroid fragments. This background can be mitigated by any of the following: titrating the antibody conjugate to a lower concentration, fixing samples with formaldehyde, or removing erythrocytes before staining (eg, gradient centrifugation or pre-lysis with wash). This background has not been observed when cells were lysed with BD FACS™ Lysing Solution (Cat. No. 349202) after staining.
  2. Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
  3. An isotype control should be used at the same concentration as the antibody of interest.
  4. 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).
  5. 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.
  6. 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.
  7. For fluorochrome spectra and suitable instrument settings, please refer to our Multicolor Flow Cytometry web page at www.bdbiosciences.com/colors.
  8. Human donor specific background has been observed in relation to the presence of anti-polyethylene glycol (PEG) antibodies, developed as a result of certain vaccines containing PEG, including some COVID-19 vaccines. We recommend use of BD Horizon Brilliant™ Stain Buffer in your experiments to help mitigate potential background. For more information visit https://www.bdbiosciences.com/en-us/support/product-notices.
  9. Species cross-reactivity detected in product development may not have been confirmed on every format and/or application.
  10. Please refer to http://regdocs.bd.com to access safety data sheets (SDS).
  11. Cy is a trademark of Global Life Sciences Solutions Germany GmbH or an affiliate doing business as Cytiva.
  12. For U.S. patents that may apply, see bd.com/patents.
569336 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.

569336 Rev. 3
Format Details
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RB780
The BD Horizon RealBlue™ 780 (RB780) Dye is part of the BD family of blue dyes. It is a tandem fluorochrome with an excitation maximum (Ex Max) at 498-nm and an emission maximum (Em Max) at 781-nm. Driven by BD innovation, RB780 can be used on both spectral and conventional cytometers and is designed to be excited by the Blue laser (488-nm) with minimal excitation by the 561-nm Yellow-Green laser. For conventional instruments equipped with a Blue laser (488-nm), RB780 can be used as an alternative to PE-Cy7 and we recommend using an optical filter centered near 780-nm (eg, a 780/60-nm bandpass filter). For spectral instruments equipped with a Blue laser (488-nm), it can be used in conjunction with PE-Cy7. RB780 is on average brighter than PE-Cy7 and has minimal spillover into Yellow-Green detectors.
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RB780
Blue 488 nm
498 nm
781 nm
569336 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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569336 Rev. 3

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