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BD Horizon™ BB515 Mouse Anti-Human TIM-3 (CD366)

Name: BB515 Mouse Anti-Human TIM-3 (CD366)
Brand: BB515 Mouse Anti-Human TIM-3 (CD366)
SKU: 565569

Clone 7D3 (RUO)

Multiparameter flow cytometric analysis of TIM-3 (CD366) expression on human peripheral blood cells. Human whole blood was stained with PE Mouse Anti-Human CD56 antibody (Cat. No. 555516/561903) and either BD Horizon™ BB515 Mouse IgG1 κ Isotype Control (Cat. No. 564416) or BD Horizon BB515 Mouse Anti-Human TIM-3 (CD366) antibody (Cat. No. 565568/565569). Erythrocytes were lysed with BD FACS Lysing Solution (Cat. No. 349202).

Left Plots: The two-parameter flow cytometric contour plots show the correlated expression patterns of TIM-3 (CD366) expression (or Ig Isotype control staining) versus side scattered light-signals (SSC-A) distinguishing monocyte (intermediate SSC-A) and lymphocyte (low SCC-A) populations.

Right Plots: The two-color flow cytometric contour plots show the correlated expression of CD56 versus TIM-3 (CD366) [or Ig Isotype control staining]. Gated events with the forward and side-light scattering characteristics of viable lymphocytes are displayed.

Flow cytometric analysis was performed using a BD™ LSR II Flow Cytometer System.

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Protocol Library

Scientific Resources

Product Details

Brand: BD Horizon™

Alternative Name: CD366; HAVCR2; TIM3; T cell immunoglobulin mucin-3; TIMD-3; KIM-3

Reactivity: Human (QC Testing)

Isotype: Mouse IgG1, κ

Immunogen: Human TIM-3

Application: Flow cytometry (Routinely Tested)

Vol. Per Test: 5 µl

Workshop Number: X

RRID: AB_2744368

Storage Buffer: Aqueous buffered solution containing ≤0.09% sodium azide.

Regulatory Status: 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 with BD Horizon™ BB515 under optimum conditions and unconjugated antibody was removed.

Recommended Assay Procedures

BD™ CompBeads can be used as surrogates to assess fluorescence spillover (Compensation). When fluorochrome conjugated antibodies are bound to 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 CompBead to ensure that BD Comp beads are appropriate for your specific cellular application.

For optimal and reproducible results, BD Horizon Brilliant Stain Buffer should be used anytime two or more BD Horizon Brilliant dyes are used in the same experiment. Fluorescent dye interactions may cause staining artifacts which may affect data interpretation. The BD Horizon Brilliant Stain Buffer was designed to minimize these interactions. 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

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).
An isotype control should be used at the same concentration as the antibody of interest.
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.
The manufacture, use, sale, offer for sale, or import of this product is subject to one or more patents or pending applications. This product, and only in the amount purchased by buyer, may be used solely for buyer’s own internal research, in a manner consistent with the accompanying product literature. No other right to use, sell or otherwise transfer (a) this product, or (b) its components is hereby granted expressly, by implication or by estoppel. Diagnostic uses require a separate license.
For fluorochrome spectra and suitable instrument settings, please refer to our Multicolor Flow Cytometry web page at www.bdbiosciences.com/colors.
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.
Please refer to http://regdocs.bd.com to access safety data sheets (SDS).
Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.

Companion Products

Stain Buffer (FBS) RUO

Size 500 ML Cat No. 554656

Stain Buffer (BSA) RUO

Size 500 ML Cat No. 554657

Brilliant Stain Buffer RUO

Size 100 Tests Cat No. 563794

Lysing Solution 10X Concentrate IVD

Size 100 ML Cat No. 349202

Lysing Buffer RUO

Size 100 ML Cat No. 555899

BB515 Mouse IgG1, κ Isotype Control RUO

Size 100 µg Cat No. 564416

View Details

565569 Rev. 2

Antibody Details

7D3

The 7D3 monoclonal antibody specifically binds to T cell immunoglobulin mucin 3 (TIM-3) which is also known as, CD366, or T-cell immunoglobulin and mucin domain-containing protein 3 (TIMD-3/TIMD3). CD366 is encoded by the HAVCR2 gene (Hepatitis A virus cellular receptor 2). CD366 is a type I transmembrane glycoprotein and belongs to the human TIM family (along with TIM-1 and TIM-4) within the immunoglobulin superfamily. CD366 is expressed on Th1, Tc1, Th17, Treg, NK T, and NK cells. CD366 is also expressed on dendritic cells, mast cells, monocytes, and macrophages. It is not expressed by Th2 and B cells. CD366 helps maintain peripheral immune tolerance and homeostasis. CD366 regulates macrophage activation and is a negative regulator of Th1 cell function. Crosslinking of cell surface CD366 by binding to Galectin-9 and/or phosphatidylserine appears to play an important role in either positively or negatively regulating leucocyte functions, such as cytokine production or the phagocytosis of apoptotic cells. CD366 may also be useful as an AML stem cell surface marker because it appears to be more highly expressed by AML leukemia stem cells than by normal bone marrow hematopoietic stem cells.

The antibody was conjugated to BD Horizon BB515 which is part of the BD Horizon Brilliant™ Blue family of dyes. With an Ex Max near 490 nm and an Em Max near 515 nm, BD Horizon BB515 can be excited by the blue laser (488 nm) laser and detected with a 530/30 nm filter. This dye has been exclusively developed by BD Biosciences and is up to seven times brighter than FITC with less spillover into the PE channel. Due to similar excitation and emission properties, BB515, FITC, and Alexa Fluor® 488 cannot be used simultaneously. It is not recommended to use BB515 in cocktails that include Streptavidin conjugates as it may cause high background.

565569 Rev. 2

Format Details

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. It is not recommended to use BB515 in cocktails that include streptavidin conjugates as it may cause high background.

Format: BB515

Excitation Max: 490 nm

Emission Max: 515 nm

565569 Rev.2

Citations & References

View product citations for antibody "565569" on CiteAb

Development References (12)

Domenig C, Zheng XX, Sabatos CA, et al. Tim-3 inhibits T helper type 1-mediated auto- and alloimmune responses and promotes immunological tolerance. Nat Immunol. 2003; 4(11):1093-1101. (Biology). View Reference
Freeman GJ, Casasnovas JM, Umetsu DT, DeKruyff RH. TIM genes: a family of cell surface phosphatidylserine receptors that regulate innate and adaptive immunity.. Immunol Rev. 2010; 235(1):172-89. (Biology). View Reference
Hafler DA, Kuchroo V. TIMs: Central regulators of immune responses. J Exp Med. 2008; 205:2699-2701. (Biology). View Reference
Jan M, Chao MP, Cha AC, et al. Prospective separation of normal and leukemic stem cells based on differential expression of TIM3, a human acute myeloid leukemia stem cell marker. Proc Natl Acad Sci U S A. 2011; 108(12):5009-5014. (Biology). View Reference
Khademi M, Illes Z, Gielen AW, et al. T Cell Ig- and mucin-domain-containing molecule-3 (TIM-3) and TIM-1 molecules are differentially expressed on human Th1 and Th2 cells and in cerebrospinal fluid-derived mononuclear cells in multiple sclerosis. J Immunol. 2004; 172(11):7169-7176. (Biology). View Reference
Lee J, Su EW, Zhu C, et al. Phosphotyrosine-dependent coupling of Tim-3 to T-cell receptor signaling pathways. Mol Cell Biol. 2011; 31(19):3963-3974. (Biology). View Reference
Lee JS, Park MJ, Park S, Lee ES. Differential expression of T cell immunoglobulin- and mucin-domain-containing molecule-3 (TIM-3) according to activity of Behcet's disease. Br J Dermatol. 2012; 65(3):220-222. (Biology). View Reference
Moorman JP, Wang JM, Zhang Y, et al. Tim-3 pathway controls regulatory and effector T cell balance during hepatitis C virus infection. J Immunol. 2012; 189(2):755-766. (Biology). View Reference
Ndhlovu LC, Lopez-Verges S, Barbour JD, et al. Tim-3 marks human natural killer cell maturation and suppresses cell-mediated cytotoxicity. Blood. 2012; 119(16):3734-3743. (Biology). View Reference
Rodriguez-Manzanet R, DeKruyff R, Kuchroo VK, Umetsu DT. The costimulatory role of TIM molecules. Immunol Rev. 2009; 229(1):259-270. (Biology). View Reference
Wang F, Wan L, Zhang C, Zheng X, Li J, Chen ZK. Tim-3-Galectin-9 pathway involves the suppression induced by CD4+CD25+ regulatory T cells. Immunobiology. 2009; 214(5):342-349. (Biology). View Reference
van de Weyer PS, Muehlfeit M, Klose C, Bonventre JV, Walz G, Kuehn EW. A highly conserved tyrosine of Tim-3 is phosphorylated upon stimulation by its ligand galectin-9. Biochem Biophys Res Commun. 2006; 351(2):571-576. (Biology). View Reference

View All (12)

565569 Rev. 2

Please refer to Support Documents for Quality Certificates

Global - Refer to manufacturer's instructions for use and related User Manuals and Technical data sheets before using this products as described

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.

Please refer to Support Documents for Quality Certificates

Global - Refer to manufacturer's instructions for use and related User Manuals and Technical data sheets before using this products as described

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.