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Purified Mouse Anti-Human CD152
Purified 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 and then stained with either Purified Mouse IgG2a, κ Isotype Control (Cat. No. 555571; dashed line histogram) or Purified Mouse Anti-Human CD152 (Cat. No. 555851; 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 and then stained with either Purified Mouse IgG2a, κ Isotype Control (Cat. No. 555571; dashed line histogram) or Purified Mouse Anti-Human CD152 (Cat. No. 555851; 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), Immunohistochemistry-frozen, Intracellular staining (flow cytometry) (Tested During Development)
0.5 mg/ml
IX 34
1493
Aqueous buffered solution containing ≤0.09% sodium azide.
RUO


Preparation And Storage

Store undiluted at 4°C. The monoclonal antibody was purified from tissue culture supernatant or ascites by affinity chromatography.

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 (1 x 10^6) with primary (unconjugated) antibody for 20-30 minutes at room temperature.

2. Add 2 mls of 1X PharM Lyse (10X PharM Lyse, Cat. No. 555899) and incubate for 10-15 minutes. Centrifuge and aspirate.

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

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

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

6. Add SAV-PE (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. 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. Sodium azide is a reversible inhibitor of oxidative metabolism; therefore, antibody preparations containing this preservative agent must not be used in cell cultures nor injected into animals. Sodium azide may be removed by washing stained cells or plate-bound antibody or dialyzing soluble antibody in sodium azide-free buffer. Since endotoxin may also affect the results of functional studies, we recommend the NA/LE (No Azide/Low Endotoxin) antibody format, if available, for in vitro and in vivo use.
  6. Species cross-reactivity detected in product development may not have been confirmed on every format and/or application.
  7. Please refer to http://regdocs.bd.com to access safety data sheets (SDS).
  8. For U.S. patents that may apply, see bd.com/patents.
555851 Rev. 9
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.

555851 Rev. 9
Format Details
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Purified
Tissue culture supernatant is purified by either protein A/G or affinity purification methods. Both methods yield antibody in solution that is free of most other soluble proteins, lipids, etc. This format provides pure antibody that is suitable for a number of downstream applications including: secondary labeling for flow cytometry or microscopy, ELISA, Western blot, etc.
Purified
555851 Rev.9
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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555851 Rev. 9

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