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BV421 Rat Anti-Mouse CD366 (TIM-3)
BV421 Rat Anti-Mouse CD366 (TIM-3)
Multiparameter flow cytometric analysis of CD366 (TIM-3) expression on activated Mouse splenic leucocytes. C57BL/6 Mouse splenocytes were cultured for 4 days in the presence of plate-bound Purified NA/LE Hamster Anti-Mouse CD3e (Cat. No. 553057/567114; 10 μg/ml for coating), soluble Purified NA/LE Hamster Anti-Mouse CD28 (Cat. No. 567110/553294; 2 μg/ml), and Purified NA/LE Rat Anti-Mouse IL-4 (Cat. No. 554432) antibodies. The cells were harvested, washed and preincubated with Purified Rat Anti-Mouse CD16/CD32 antibody (Mouse BD Fc Block™) [Cat. No. 553141/553142]. The cells were then stained with PE Rat Anti-Mouse CD8a antibody (Cat. No. 553033/553032/561095) and with either BD Horizon™ BV421 Rat IgG2a, κ Isotype Control (Cat. No. 562602; Left Plot) or BD Horizon™ BV421 Rat Anti-Mouse CD366 (TIM-3) antibody (Cat. No. 568798/568799; Right Plot) at 0.5 µg/test. BD Via-Probe™ Cell Viability 7-AAD Solution (Cat. No. 555815/555816) was added to cells right before analysis.  The bivariate pseudocolor density plot showing the expression of CD366 (TIM-3) [or Ig Isotype control staining] versus CD8a was derived from gated events with the forward and side light-scatter characteristics of viable (7-AAD-negative) splenic lymphocytes. Flow cytometry and data analysis were performed a BD LSRFortessa™ X-20 Cell Analyzer System and FlowJo™ software. Data shown on this Technical Data Sheet are not lot specific.
Multiparameter flow cytometric analysis of CD366 (TIM-3) expression on activated Mouse splenic leucocytes. C57BL/6 Mouse splenocytes were cultured for 4 days in the presence of plate-bound Purified NA/LE Hamster Anti-Mouse CD3e (Cat. No. 553057/567114; 10 μg/ml for coating), soluble Purified NA/LE Hamster Anti-Mouse CD28 (Cat. No. 567110/553294; 2 μg/ml), and Purified NA/LE Rat Anti-Mouse IL-4 (Cat. No. 554432) antibodies. The cells were harvested, washed and preincubated with Purified Rat Anti-Mouse CD16/CD32 antibody (Mouse BD Fc Block™) [Cat. No. 553141/553142]. The cells were then stained with PE Rat Anti-Mouse CD8a antibody (Cat. No. 553033/553032/561095) and with either BD Horizon™ BV421 Rat IgG2a, κ Isotype Control (Cat. No. 562602; Left Plot) or BD Horizon™ BV421 Rat Anti-Mouse CD366 (TIM-3) antibody (Cat. No. 568798/568799; Right Plot) at 0.5 µg/test. BD Via-Probe™ Cell Viability 7-AAD Solution (Cat. No. 555815/555816) was added to cells right before analysis.  The bivariate pseudocolor density plot showing the expression of CD366 (TIM-3) [or Ig Isotype control staining] versus CD8a was derived from gated events with the forward and side light-scatter characteristics of viable (7-AAD-negative) splenic lymphocytes. Flow cytometry and data analysis were performed a BD LSRFortessa™ X-20 Cell Analyzer System and FlowJo™ software. Data shown on this Technical Data Sheet are not lot specific.
Product Details
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BD Horizon™
Cd366; TIM-3; Tim3; TIMD-3; Timd3; HAVcr-2; Havcr2
Mouse (QC Testing)
Rat SD, also known as Sprague-Dawley (outbred) IgG2a, κ
Mouse TIM-3 Recombinant Protein
Flow cytometry (Routinely Tested)
0.2 mg/ml
171285
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.

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.

   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).

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. For fluorochrome spectra and suitable instrument settings, please refer to our Multicolor Flow Cytometry web page at www.bdbiosciences.com/colors.
  6. BD Horizon Brilliant Violet 421 is covered by one or more of the following US patents: 8,158,444; 8,362,193; 8,575,303; 8,354,239.
  7. 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.
  8. Please refer to http://regdocs.bd.com to access safety data sheets (SDS).
Antibody Details
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RMT3-23

The RMT3-23 monoclonal antibody specifically recognizes CD366 which is also known as TIM-3 (T-cell immunoglobulin and mucin domain-containing 3,  T-cell immunoglobulin mucin receptor 3, or T-cell membrane protein 3). CD366 (TIM-3) is ~31 kDa type I transmembrane glycoprotein encoded by Havcr2 (Hepatitis A virus cellular receptor 2) that belongs to the TIM family within the immunoglobulin superfamily (IgSF). CD366 (TIM-3) is comprised of one Ig-like V-type domain followed by a serine/threonine-rich mucin stalk region in its extracellular region, a transmembrane segment, and a tyrosine phosphorylation motif in its cytoplasmic tail. CD366 (TIM-3) expression is upregulated on subpopulations of activated myeloid cells including macrophages, monocytes, dendritic cells (DC), microglia, mast cells as well as on Type-1 CD4+ (Th1-like) T cells, cytotoxic CD8+ T cells, regulatory T cells (Treg), and natural killer (NK) cells. CD366 (TIM-3) functions as an inhibitory receptor that helps maintain immunological homeostasis and self-tolerance. It may also serve an immune checkpoint molecule that inhibits antitumor immunity and promotes T cell exhaustion. Crosslinking of cell surface CD366 (TIM-3) by Galectin-9 binding downregulates Th1-like and CD8+ T cell responses and can promote Treg or myeloid-derived suppressor cells. CD366 (TIM-3) enables DC to bind phosphatidyl serine expressed by apoptotic cells, phagocytize these cells to suppress inflammation and promote antigen cross-presentation. CD366 (TIM-3) can also bind to high mobility group protein B1 (HMGB1) and inhibit stimulation of the immune response to nucleic acids released by dying tumor cells. RMT3-23 reportedly recognizes CD366 (TIM-3) derived from either BALB/c or C57BL/6 mice that have different Havcr2 polymorphisms.

Format Details
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BV421
The BD Horizon Brilliant Violet™ 421 (BV421) Dye is part of the BD Horizon Brilliant Violet™ family of dyes. This polymer-technology based dye has an excitation maximum (Ex Max) of 407-nm and an emission maximum (Em Max) at 423-nm. Driven by BD innovation, BV421 is designed to be excited by the violet laser (405-nm) and detected using an optical filter centered near 420-nm (e.g., a 431/28-nm or 450/50-nm bandpass filter). BV421 is an ideal alternative for V450 as it is approximately ten times brighter with less spillover into the BV510/V500 detector. Please ensure that your instrument’s configurations (lasers and optical filters) are appropriate for this dye.
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BV421
Violet 405 nm
407 nm
423 nm
Citations & References
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View product citations for antibody "568799" on CiteAb

Development References (10)

  1. Anderson AC, Joller N, Kuchroo VK. Lag-3, Tim-3, and TIGIT: Co-inhibitory Receptors with Specialized Functions in Immune Regulation.. Immunity. 2016; 44(5):989-1004. (Biology). View Reference
  2. Anderson AC, Xiao S, Kuchroo VK. Tim protein structures reveal a unique face for ligand binding.. Immunity. 2007; 26(3):273-5. (Biology). View Reference
  3. Chiba S, Baghdadi M, Akiba H, et al. Tumor-infiltrating DCs suppress nucleic acid-mediated innate immune responses through interactions between the receptor TIM-3 and the alarmin HMGB1.. Nat Immunol. 2012; 13(9):832-42. (Biology). View Reference
  4. Nakae S, Iikura M, Suto H, et al. TIM-1 and TIM-3 enhancement of Th2 cytokine production by mast cells.. Blood. 2007; 110(7):2565-8. (Clone-specific: Flow cytometry). View Reference
  5. Nakayama M, Akiba H, Takeda K, et al. Tim-3 mediates phagocytosis of apoptotic cells and cross-presentation. Blood. 2009; 113(16):3821-3830. (Clone-specific). View Reference
  6. Ngiow SF, von Scheidt B, Akiba H, Yagita H, Teng MW, Smyth MJ. Anti-TIM3 antibody promotes T cell IFN-γ-mediated antitumor immunity and suppresses established tumors.. Cancer Res. 2011; 71(10):3540-51. (Clone-specific: Flow cytometry). View Reference
  7. Oikawa T1, Kamimura Y, Akiba H, et al. Preferential involvement of Tim-3 in the regulation of hepatic CD8+ T cells in murine acute graft-versus-host disease.. J Immunol. 2006; 177(7):4281-4287. (Immunogen: Flow cytometry, Immunohistochemistry, In vivo exacerbation). View Reference
  8. Sakuishi K, Ngiow SF, Sullivan JM, et al. TIM3+FOXP3+ regulatory T cells are tissue-specific promoters of T-cell dysfunction in cancer.. Oncoimmunology. 2013; 2(4):e23849. (Clone-specific: Flow cytometry). View Reference
  9. Veenstra RG, Taylor PA, Zhou Q, et al. Contrasting acute graft-versus-host disease effects of Tim-3/galectin-9 pathway blockade dependent upon the presence of donor regulatory T cells.. Blood. 2012; 120(3):682-90. (Biology). View Reference
  10. Zhu C, Anderson AC, Schubart A, et al. The Tim-3 ligand galectin-9 negatively regulates T helper type 1 immunity.. Nat Immunol. 2005; 6(12):1245-52. (Methodology). View Reference
View All (10) View Less

 

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