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67761S 100 µl (50 tests) $329.00


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H Endogenous Rabbit IgG

Flow Cytometry

Flow cytometric analysis of Jurkat cells (blue) and primary CD4+ T cells (green) using TIM-3 (D5D5R™) XP® Rabbit mAb (PE Conjugate). Controls were run on Jurkat cells (orange) and primary CD4+ T cells (red) using concentration-matched Rabbit (DA1E) mAb IgG XP® Isotype Control (PE Conjugate) #5742. CD4+ T cells were purified from human blood and stimulated for 9 days using beads coated with CD3 and CD28 antibodies in the presence of Human Interleukin-2 (hIL-2) #8907 (6.7 ng/ml).

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Flow Cytometry Protocol for TIM-3 (D5D5R) XP® Rabbit mAb (PE Conjugate) #67761

A. Solutions and Reagents

  1. 1X Phosphate Buffered Saline (PBS): Dissolve 8 g NaCl, 0.2 g KCl, 1.44 g Na2HPO4, and 0.24 g KH2PO4 in 800 ml distilled water (dH2O). Adjust the pH to 7.4 with HCl and the volume to 1 liter with dH2O. Store at room temperature.
  2. Incubation Buffer: Dissolve 0.5 g bovine serum albumin (BSA) in 100 ml 1X PBS. Store at 4°C.

B. Immunostaining

NOTE: Account for isotype matched controls for monoclonal antibodies or species matched IgG for polyclonal antibodies. Count cells using a hemacytometer or alternative method.

  1. Collect cells by centrifugation and aspirate supernatant.
  2. Aliquot 0.5–1 x 106 cells into each assay tube (by volume).
  3. Add 2–3 ml Incubation Buffer to each tube and rinse by centrifugation. Repeat.
  4. Resuspend cells in 100 µl Incubation Buffer per assay tube.
  5. Block in Incubation Buffer for 10 minutes on ice.
  6. Add the conjugated antibody at the appropriate dilution to the assay tubes (see individual antibody data sheet for the dilution recommendations).
  7. Incubate for 1 hour on ice.
  8. Rinse as before in Incubation Buffer by centrifugation.
  9. Resuspend cells in 0.5 ml PBS and analyze on flow cytometer.

revised April 2016

Protocol Id: 1104

Product Usage Information

Application Dilutions
Flow Cytometry 1:50

Storage: Supplied in PBS (pH 7.2), less than 0.1% sodium azide and 2 mg/ml BSA. Store at 4°C. Do not aliquot the antibody. Protect from light. Do not freeze.

Specificity / Sensitivity

TIM-3 (D5D5R™) XP® Rabbit mAb (PE Conjugate) recognizes endogenous levels of total TIM-3 protein.

Species Reactivity: Human

Source / Purification

Monoclonal antibody is produced by immunizing animals with recombinant protein specific to the extracellular domain of human TIM-3 protein.

Product Description

This Cell Signaling Technology antibody is conjugated to phycoerythrin (PE) and tested in-house for direct flow cytometry analysis in human cells. This antibody is expected to exhibit the same species cross-reactivity as the unconjugated TIM-3 (D5D5R™) XP® Rabbit mAb #45208.

T cell Ig- and mucin-domain-containing molecules (TIMs) are a family of transmembrane proteins expressed by various immune cells. TIM-3 is an inhibitory molecule that is induced following T cell activation (1-3 ). TIM-3 is expressed by exhausted T cells in the settings of chronic infection and cancer (4,5), and tumor-infiltrating T cells that coexpress PD-1 and TIM-3 exhibit the most severe exhausted phenotype (5). Tumor-infiltrating dendritic cells (DCs) also express TIM-3. TIM-3 expression on DCs was found to suppress innate immunity by reducing the immunogenicity of nucleic acids released by dying tumor cells (6). Research studies show that heterodimerization of TIM-3 with CEACAM-1 is critical for the inhibitory function of TIM-3, and co-blockade of TIM-3 and CEACAM-1 enhanced antitumor responses in a mouse model of colorectal cancer (7). In addition, blockade of TIM-3 in mouse models of autoimmunity enhanced the severity of disease (1). Finally, binding of Galectin-9 to TIM-3 expressed by Th1 cells induces T cell death (8).

1.  Monney, L. et al. (2002) Nature 415, 536-41.

2.  Sánchez-Fueyo, A. et al. (2003) Nat Immunol 4, 1093-101.

3.  Sabatos, C.A. et al. (2003) Nat Immunol 4, 1102-10.

4.  Jones, R.B. et al. (2008) J Exp Med 205, 2763-79.

5.  Sakuishi, K. et al. (2010) J Exp Med 207, 2187-94.

6.  Chiba, S. et al. (2012) Nat Immunol 13, 832-42.

7.  Huang, Y.H. et al. (2015) Nature 517, 386-90.

8.  Zhu, C. et al. (2005) Nat Immunol 6, 1245-52.

Entrez-Gene Id 84868
Swiss-Prot Acc. Q8TDQ0

For Research Use Only. Not For Use In Diagnostic Procedures.
Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
XP is a registered trademark of Cell Signaling Technology, Inc.

TIM-3 (D5D5R™) XP® Rabbit mAb (PE Conjugate)