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Purified NA/LE Mouse Anti-Human CD152
Purified NA/LE Mouse Anti-Human CD152
Flow cytometric analysis of CD152 expression on stimulated Human peripheral mononuclear cells. Peripheral blood mononuclear cells were stimulated with Concanavalin A for 3 days, then stained with either Purified NA/LE Mouse IgG2a, κ Isotype Control (Cat. No. 554645; dashed line histogram) or Purified NA/LE Mouse Anti-Human CD152 (Cat. No. 555850; solid line histogram), followed by Biotin Goat Anti-Mouse Ig (Multiple Adsorption) (Cat. No. 553999) and PE Streptavidin (Cat. No. 554061). The fluorescence histograms were derived from gated events with the forward and side light-scattering characteristics of viable activated cells. Flow cytometry was performed on a BD FACScan™ system.
Flow cytometric analysis of CD152 expression on stimulated Human peripheral mononuclear cells. Peripheral blood mononuclear cells were stimulated with Concanavalin A for 3 days, then stained with either Purified NA/LE Mouse IgG2a, κ Isotype Control (Cat. No. 554645; dashed line histogram) or Purified NA/LE Mouse Anti-Human CD152 (Cat. No. 555850; solid line histogram), followed by Biotin Goat Anti-Mouse Ig (Multiple Adsorption) (Cat. No. 553999) and PE Streptavidin (Cat. No. 554061). The fluorescence histograms were derived from gated events with the forward and side light-scattering characteristics of viable activated cells. Flow cytometry was performed on a BD FACScan™ system.
Product Details
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BD Pharmingen™
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), Functional assay (Tested During Development)
1.0 mg/ml
IX 34
1493
AB_396173
No azide/low endotoxin: Aqueous buffered solution containing no preservative, 0.2 µm sterile filtered. Endotoxin level is ≤0.01 EU/µg (≤0.001 ng/µg) of protein as determined by the LAL assay.
RUO


Preparation And Storage

Store undiluted at 4°C. The monoclonal antibody was purified from tissue culture supernatant or ascites by affinity chromatography. This preparation contains no preservatives, thus it should be handled under aseptic conditions.

Recommended Assay Procedures

For flow cytometric applications, a three step labeling procedure is recommended for amplifying signal. Suggested protocol for 3-step staining using concanavalin-A-stimulated peripheral blood mononuclear cells method:

1. Incubate 100 µl concanavalin-A-stimulated 3-day peripheral blood mononuclear cells (1x10^6) with primary (unconjugated) antibody for 20-30 minutes at room temperature.

2. Add 2 mls of 1X lysing buffer (10X Pharm Lyse Lysing Buffer, Cat. No. 555899) and incubate for 10-15 minutes. Centrifuge and aspirate.

3. Wash once with PBS/0.1% sodium azide/1% heat-activated fetal bovine serum (PBS-FBS). Centrifuge and aspirate.

4. Add biotinylated goat anti-mouse Ig  (Cat. No. 553999) and incubate for 20-30 minutes at room temperature.

5. Wash once with PBS-FBS. Centrifuge and aspirate.

6. Add PE-Streptavidin (Cat. No. 554061) and incubate for 20-30 minutes in the dark at room temperature.

7. Wash once with PBS-FBS. Centrifuge and aspirate. Resuspend in 0.5 ml of PBS-FBS and analyze by flow cytometry.

Product Notices

  1. Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
  2. Since applications vary, each investigator should titrate the reagent to obtain optimal results.
  3. An isotype control should be used at the same concentration as the antibody of interest.
  4. Species cross-reactivity detected in product development may not have been confirmed on every format and/or application.
  5. Please refer to http://regdocs.bd.com to access safety data sheets (SDS).
  6. For U.S. patents that may apply, see bd.com/patents.
555850 Rev. 12
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.

555850 Rev. 12
Format Details
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NA/LE
NA/LE refers to the culture and purification methods and buffer used to produce purified antibodies with no azide and low endotoxin: Aqueous buffered solution containing no preservative, 0.2µm sterile filtered. Endotoxin level is ≤0.01 EU/µg (≤0.001 ng/µg) of protein as determined by the LAL assay.NA/LE are perfectly suited to be used in culture or in vivo (for nonhuman studies) for functional assays — blocking, neutralizing, activation or depletion — where the presence of azide may damage cells or exogenous endotoxin may signal or activate cells.
NA/LE
555850 Rev.12
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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555850 Rev. 12

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