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BB515 Mouse Anti-Human GITR (CD357)
BB515 Mouse Anti-Human GITR (CD357)
Multiparameter flow cytometric analysis of GITR (CD357) expression on human peripheral blood lymphocytes. PBMC were cultured for 3 days in medium without (Unstimulated; Left Plot) or with Phytohemagglutinin-M (PHA-M Stimulated; 1.5% v/v; Right Plot). Cells were stained with APC Mouse Anti-Human CD4 antibody (Cat. No. 561841) and with either BD Horizon™ BB515 IgG2b, κ Isotype Control (Cat. No. 564510; no staining, data not shown) or BD Horizon™ Mouse Anti-Human GITR (CD357) antibody (Cat. No. 567781/567782). BD Via-Probe™ Cell Viability 7-AAD Solution (Cat. No. 555815/555816) was added to cells right before analysis. The bivariate pseudocolor density plots showing the correlated expression of GITR (CD357) versus CD4 were derived from gated events with the forward and side light-scatter characteristics of viable (7-AAD-negative) human peripheral blood lymphocytes. Multiparameter flow cytometric analysis of GITR (CD357) expression on human CD4+ peripheral blood lymphocytes. PBMC were stained with APC Mouse Anti-Human CD4 (Cat. No. 561841) and BD Horizon™ BV421 Mouse Anti-Human CD25 (Cat. No. 562442) antibodies and with either BD Horizon™ BB515 IgG2b, κ Isotype Control (Cat. No. 564510; dashed line histograms) or BD Horizon™ BB515 Mouse Anti-Human GITR (CD357) antibody (Cat. No. 567781/567782); solid line histograms). BD Via-Probe™ Cell Viability 7-AAD Solution was added to cells right before analysis. Histograms showing the expression of GITR (CD357) [or Ig Isotype control staining] were derived from CD4+CD25-negative (Left Plot) or CD4+CD25-bright positive (Right Plot) gated events with the forward and side light-scatter characteristics of viable (7-AAD negative) lymphocytes. Flow cytometry and data analysis were performed using a BD LSRFortessa™ X-20 Cell Analyzer System and FlowJo™ software.
Multiparameter flow cytometric analysis of GITR (CD357) expression on human peripheral blood lymphocytes. PBMC were cultured for 3 days in medium without (Unstimulated; Left Plot) or with Phytohemagglutinin-M (PHA-M Stimulated; 1.5% v/v; Right Plot). Cells were stained with APC Mouse Anti-Human CD4 antibody (Cat. No. 561841) and with either BD Horizon™ BB515 IgG2b, κ Isotype Control (Cat. No. 564510; no staining, data not shown) or BD Horizon™ Mouse Anti-Human GITR (CD357) antibody (Cat. No. 567781/567782). BD Via-Probe™ Cell Viability 7-AAD Solution (Cat. No. 555815/555816) was added to cells right before analysis. The bivariate pseudocolor density plots showing the correlated expression of GITR (CD357) versus CD4 were derived from gated events with the forward and side light-scatter characteristics of viable (7-AAD-negative) human peripheral blood lymphocytes. Multiparameter flow cytometric analysis of GITR (CD357) expression on human CD4+ peripheral blood lymphocytes. PBMC were stained with APC Mouse Anti-Human CD4 (Cat. No. 561841) and BD Horizon™ BV421 Mouse Anti-Human CD25 (Cat. No. 562442) antibodies and with either BD Horizon™ BB515 IgG2b, κ Isotype Control (Cat. No. 564510; dashed line histograms) or BD Horizon™ BB515 Mouse Anti-Human GITR (CD357) antibody (Cat. No. 567781/567782); solid line histograms). BD Via-Probe™ Cell Viability 7-AAD Solution was added to cells right before analysis. Histograms showing the expression of GITR (CD357) [or Ig Isotype control staining] were derived from CD4+CD25-negative (Left Plot) or CD4+CD25-bright positive (Right Plot) gated events with the forward and side light-scatter characteristics of viable (7-AAD negative) lymphocytes. Flow cytometry and data analysis were performed using a BD LSRFortessa™ X-20 Cell Analyzer System and FlowJo™ software.
製品詳細
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BD Horizon™
GITR-D; TNFRSF18; activation-inducible TNFR family receptor; AITR
Human (QC Testing)
Mouse BALB/c IgG2b, κ
Human GITR Recombinant Protein
Flow cytometry (Routinely Tested)
5 µl
8784
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.

推奨アッセイ手順

     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. 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.
  3. For fluorochrome spectra and suitable instrument settings, please refer to our Multicolor Flow Cytometry web page at www.bdbiosciences.com/colors.
  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. An isotype control should be used at the same concentration as the antibody of interest.
  6. 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.
  7. Please refer to http://regdocs.bd.com to access safety data sheets (SDS).
  8. Alexa Fluor™ is a trademark of Life Technologies Corporation.
567782 Rev. 1
抗体の詳細
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V27-580

The V27-580 monoclonal antibody specifically binds to GITR (Glucocorticoid-Induced Tumor necrosis factor Receptor), a member of the tumor necrosis factor receptor (TNFR) superfamily that is designated TNFRSF18. In the human, GITR is expressed at low levels in peripheral blood T cells, bone marrow, thymus, spleen, and lymph nodes and is up-regulated upon antigen stimulation or by treatment with anti-CD3 plus anti-CD28. GITR is also reported to be constitutively expressed on Treg cells. GITR's ligand (GITRL) is a member of the TNF superfamily, is designated TNFSF18, and is expressed on antigen presenting cells. The GITR cytoplasmic domain has striking homology with the cytoplasmic domains of the co-stimulatory receptors CD137 (4-1BB), CD134 (OX40) and CD27. GITR signaling is mediated by signaling adaptors, TNFR-associated factors (TRAFs), that affect signaling pathways (eg, Erk, JNK, MAPK and NF-κB) to enhance T-cell survival and cytokine production. The effects of GITR signaling upon the dynamic and interconnected roles of effector and regulatory T lymphocytes in the immune response are under investigation.

567782 Rev. 1
フォーマットの詳細
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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.
altImg
BB515
Blue 488 nm
490 nm
515 nm
567782 Rev.1
引用&参考文献
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Development References (7)

  1. Clouthier DL, Watts TH. Cell-specific and context-dependent effects of GITR in cancer, autoimmunity, and infection.. Cytokine Growth Factor Rev. 2014; 25(2):91-106. (Biology). View Reference
  2. Kwon B, Yu KY, Ni J, et al. Identification of a novel activation-inducible protein of the tumor necrosis factor receptor superfamily and its ligand.. J Biol Chem. 1999; 274(10):6056-61. (Biology). View Reference
  3. Li Z, Mahesh SP, Kim BJ, Buggage RR, Nussenblatt RB. Expression of glucocorticoid induced TNF receptor family related protein (GITR) on peripheral T cells from normal human donors and patients with non-infectious uveitis.. J Autoimmun. 2003; 21(1):83-92. (Biology). View Reference
  4. Nocentini G, Giunchi L, Ronchetti S, et al. A new member of the tumor necrosis factor / nerve growth factor receptor family inhibits T cell receptor-induced apoptosis... Proc Natl Acad Sci U S A. 1997; 94(12):6216-6221. (Biology: Flow cytometry). View Reference
  5. Pedroza-Gonzalez A, Zhou G, Singh SP, et al. GITR engagement in combination with CTLA-4 blockade completely abrogates immunosuppression mediated by human liver tumor-derived regulatory T cells ex vivo.. Oncoimmunology. 2015; 4(12):e1051297. (Biology: Flow cytometry). View Reference
  6. Placke T, Kopp HG, Salih HR. Glucocorticoid-induced TNFR-related (GITR) protein and its ligand in antitumor immunity: functional role and therapeutic modulation.. Clin Dev Immunol. 2010; 2010:239083. (Biology). View Reference
  7. Shevach EM, Stephens GL. The GITR-GITRL interaction: co-stimulation or contrasuppression of regulatory activity?. Nat Rev Immunol. 2006; 6(8):613-8. (Biology). View Reference
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567782 Rev. 1

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