Flow cytometric analysis of PANC-1 cells (blue) and U-937 cells (green) using Galectin-9 (D9R4A) XP® Rabbit mAb (Alexa Fluor® 647 Conjugate) (solid lines) or a concentration-matched Rabbit (DA1E) mAb IgG XP® Isotype Control (Alexa Fluor® 647 Conjugate) #2985 (dashed lines).
This Cell Signaling Technology antibody is conjugated to Alexa Fluor® 647 fluorescent dye and tested in-house for direct flow cytometric analysis in human cells. This antibody is expected to exhibit the same species cross-reactivity as the unconjugated Galectin-9 (D9R4A) XP® Rabbit mAb #54330.
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.
All reagents required for this protocol may be efficiently purchased together in our Intracellular Flow Cytometry Kit (Methanol) #13593, or individually using the catalog numbers listed below.
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.
NOTE: When including fluorescent cellular dyes in your experiment (including viability dyes, DNA dyes, etc.), please refer to the dye product page for the recommended protocol. Visit www.cellsignal.com for a full listing of cellular dyes validated for use in flow cytometry.
NOTE: Adherent cells or tissue should be dissociated and in single-cell suspension prior to fixation.
NOTE: Optimal centrifugation conditions will vary depending upon cell type and reagent volume. Generally, 150-300g for 1-5 minutes will be sufficient to pellet the cells.
NOTE: If using whole blood, lyse red blood cells and wash by centrifugation prior to fixation.
NOTE: Antibodies targeting CD markers or other extracellular proteins may be added prior to fixation if the epitope is disrupted by formaldehyde and/or methanol. The antibodies will remain bound to the target of interest during the fixation and permeabilization process. However, note that some fluorophores (including PE and APC) are damaged by methanol and thus should not be added prior to permeabilization. Conduct a small-scale experiment if you are unsure.
NOTE: Count cells using a hemocytometer or alternative method.
posted July 2009
revised June 2020
Protocol Id: 407
Galectin-9 (D9R4A) XP® Rabbit mAb (Alexa Fluor® 647 Conjugate) recognizes endogenous levels of total galectin-9 protein.
Monoclonal antibody is produced by immunizing animals with recombinant human galectin-9 protein.
Galectins are a family of β-galactose binding proteins that are characterized by an affinity for poly-N-acetyllactosamine-enriched glycoconjugates and a carbohydrate-binding site (1,2). Members of the galectin family have been implicated in a variety of biological functions, including cell adhesion (3), growth regulation (4), cytokine production (5), T-cell apoptosis (6), and immune responses (7).
Galectin-9 is induced by proinflammatory stimuli, including IFN-γ, TNF-α, and TLR ligands, and regulates various immune responses through interaction with its ligand TIM-3 (8, 9). Binding of galectin-9 to TIM-3 expressed by Th1 CD4 T cells resulted in T cell death (9). On the other hand, galectin-9 treatment of tumor-bearing mice increased the number of IFN-γ-producing TIM-3+ CD8 T cells and TIM-3+ dendritic cells (10). Transgenic overexpression of either TIM-3 or galectin-9 in mice led to an increase in cells with a myeloid-derived suppressor cell phenotype and inhibition of immune responses (11). CD44 is also proposed to be a receptor for galectin-9, and interaction of galectin-9 with CD44 expressed by induced regulatory T (iTreg) cells enhanced the stability of function of iTreg cells. In addition, galectin-9 was recently demonstrated to bind Dectin-1 expressed by pancreatic ductal adenocarcinoma-infiltrating macrophages, resulting in tolerogenic macrophage reprogramming and suppression of anti-tumor immunity. Increased galectin-9 expression has been observed in several cancer types, including lung, liver, breast, and kidney (12). Alternative splicing of the galectin-9 transcript leads to several isoforms (13).
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Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
XP is a registered trademark of Cell Signaling Technology, Inc.
Alexa Fluor is a registered trademark of Life Technologies Corporation.
The Alexa Fluor dye conjugates in this product are sold under license from Life Technologies Corporation, for research use only excluding use in combination with DNA microarrays and high content screening (HCS).
This product is provided under an intellectual property license from Life Technologies Corporation. The transfer of this product is conditioned on the buyer using the purchased product solely in research conducted by the buyer, excluding contract research or any fee for service research, and the buyer must not (1) use this product or its components for (a) diagnostic, therapeutic or prophylactic purposes; (b) testing, analysis or screening services, or information in return for compensation on a per-test basis; or (c) manufacturing or quality assurance or quality control, and/or (2) sell or transfer this product or its components for resale, whether or not resold for use in research. For information on purchasing a license to this product for purposes other than as described above, contact Life Technologies Corporation, 5791 Van Allen Way, Carlsbad, CA 92008 USA or [email protected]