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APC Mouse Anti-Mouse CD366 (TIM-3)
APC Mouse Anti-Mouse CD366 (TIM-3)
Flow cytometric analysis of CD366 (TIM-3) expressed on activated mouse splenocytes. BALB/c splenic leucocytes were cultured for 4 days in the presence of plate-bound Purified NA/LE Hamster Anti-Mouse CD3e (Cat. No. 553057; 10 μg/ml for coating), soluble Purified NA/LE Hamster Anti-Mouse CD28 (Cat. No. 553294; 2 μg/ml), and Purified NA/LE Rat Anti-Mouse IL-4 (Cat. No. 554432) antibodies. The cells were harvested, preincubated with Purified Rat Anti-Mouse CD16/CD32 antibody (Mouse BD Fc Block™) (Cat. No. 553141/553142) and stained with BD Horizon™ BUV395 Rat Anti-Mouse CD8a antibody (Cat. No. 563786/565968) and with either APC Mouse IgG1, κ Isotype Control (Cat. No. 554681; Left Plot) or APC Mouse Anti-Mouse CD366 (TIM-3) antibody (Cat. No. 567164; Right Plot) at 0.5 μg per test. BD Via-Probe™ Cell Viability 7-AAD Solution (Cat. No. 555815/555816) was added to cells right before analysis. A bivariate pseudocolor density plot showing the correlated 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) cells. Flow cytometric analysis was performed using a BD LSRFortessa™ Cell Analyzer System. Data shown on this Technical Data Sheet are not lot specific.
Flow cytometric analysis of CD366 (TIM-3) expressed on activated mouse splenocytes. BALB/c splenic leucocytes were cultured for 4 days in the presence of plate-bound Purified NA/LE Hamster Anti-Mouse CD3e (Cat. No. 553057; 10 μg/ml for coating), soluble Purified NA/LE Hamster Anti-Mouse CD28 (Cat. No. 553294; 2 μg/ml), and Purified NA/LE Rat Anti-Mouse IL-4 (Cat. No. 554432) antibodies. The cells were harvested, preincubated with Purified Rat Anti-Mouse CD16/CD32 antibody (Mouse BD Fc Block™) (Cat. No. 553141/553142) and stained with BD Horizon™ BUV395 Rat Anti-Mouse CD8a antibody (Cat. No. 563786/565968) and with either APC Mouse IgG1, κ Isotype Control (Cat. No. 554681; Left Plot) or APC Mouse Anti-Mouse CD366 (TIM-3) antibody (Cat. No. 567164; Right Plot) at 0.5 μg per test. BD Via-Probe™ Cell Viability 7-AAD Solution (Cat. No. 555815/555816) was added to cells right before analysis. A bivariate pseudocolor density plot showing the correlated 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) cells. Flow cytometric analysis was performed using a BD LSRFortessa™ Cell Analyzer System. Data shown on this Technical Data Sheet are not lot specific.
Product Details
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BD Pharmingen™
Cd366; T-cell membrane protein; Tim3; TIMD-3; Timd3; HAVcr-2; Havcr2;
Mouse (QC Testing)
Mouse IgG1, κ
Mouse TIM-3 Recombinant Protein
Flow cytometry (Routinely Tested)
0.2 mg/ml
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 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.

Product Notices

  1. Since applications vary, each investigator should titrate the reagent to obtain optimal results.
  2. An isotype control should be used at the same concentration as the antibody of interest.
  3. 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.
  4. This APC-conjugated reagent can be used in any flow cytometer equipped with a dye, HeNe, or red diode laser.
  5. For fluorochrome spectra and suitable instrument settings, please refer to our Multicolor Flow Cytometry web page at www.bdbiosciences.com/colors.
  6. Please refer to http://regdocs.bd.com to access safety data sheets (SDS).
  7. Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
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Antibody Details
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5D12/TIM-3

The 5D12 monoclonal antibody specifically recognizes CD366 which is also known as TIM-3 (T-cell immunoglobulin and mucin domain-containing 3) or T-cell membrane protein 3. TIM-3 is encoded by Havcr2 (hepatitis A virus cellular receptor 2). TIM-3 is a type I transmembrane glycoprotein that belongs to the human TIM family within the immunoglobulin superfamily, having one Ig-like V-type domain in its extracellular region. TIM-3 is expressed on activated monocytes, macrophages, dendritic cells, microglia, and mast cells. It is also expressed on type-1 CD4+ (Th1-like) T cells, CD8+ T cytotoxic cells, regulatory T cells (Treg), and natural killer (NK) cells. TIM-3 functions as an inhibitory receptor which helps cells maintain immunological homeostasis and self tolerance. Crosslinking of cell surface TIM-3 by Galectin-9 binding downregulates Th1-like and CD8+ T cell responses and can promote Treg or myeloid-derived suppressor cells. TIM-3 enables dendritic cells to bind phosphatidyl serine expressed by apoptotic cells and to phagocytize these cells to quell potential inflammation. TIM-3 also binds to the alarmin HMGB1 thereby preventing Toll-like receptor (TLR) by released tumor cell DNA.

        

567164 Rev. 1
Format Details
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APC
Allophycocyanin (APC), is part of the BD family of phycobiliprotein dyes. This fluorochrome is a multimeric fluorescent phycobiliprotein with excitation maximum (Ex Max) of 651 nm and an emission maximum (Em Max) at 660 nm. APC is designed to be excited by the Red (627-640 nm) laser and detected using an optical filter centered near 660 nm (e.g., a 660/20 nm bandpass filter). Please ensure that your instrument’s configurations (lasers and optical filters) are appropriate for this dye.
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APC
Red 627-640 nm
651 nm
660 nm
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Citations & References
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Development References (4)

  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. 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. (Biology). View Reference
  3. Phong BL, Avery L, Sumpter TL, et al. Tim-3 enhances FcεRI-proximal signaling to modulate mast cell activation.. J Exp Med. 2015; 212(13):2289-304. (Clone-specific: Flow cytometry). View Reference
  4. 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. (Clone-specific: Flow cytometry). View Reference
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567164 Rev. 1

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