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53500
Galectin-9 (D9R4A) XP® Rabbit mAb (Alexa Fluor® 488 Conjugate)
Antibody Conjugates

Galectin-9 (D9R4A) XP® Rabbit mAb (Alexa Fluor® 488 Conjugate) #53500

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Flow cytometric analysis of Panc-1 cells (blue) and U-937 cells (green) using Galectin-9 (D9R4A) XP® Rabbit mAb (Alexa Fluor® 488 Conjugate) (solid lines) or a concentration-matched Rabbit (DA1E) mAb IgG XP® Isotype Control (Alexa Fluor® 488 Conjugate) #2975 (dashed lines).

To Purchase # 53500S
Product # Size Price
53500S
100 µl  (50 tests) $ 348

Supporting Data

REACTIVITY H
SENSITIVITY Endogenous
MW (kDa)
Isotype Rabbit IgG

Application Key:

  • W-Western
  • IP-Immunoprecipitation
  • IHC-Immunohistochemistry
  • ChIP-Chromatin Immunoprecipitation
  • IF-Immunofluorescence
  • F-Flow Cytometry
  • E-P-ELISA-Peptide

Species Cross-Reactivity Key:

  • H-Human
  • M-Mouse
  • R-Rat
  • Hm-Hamster
  • Mk-Monkey
  • Mi-Mink
  • C-Chicken
  • Dm-D. melanogaster
  • X-Xenopus
  • Z-Zebrafish
  • B-Bovine
  • Dg-Dog
  • Pg-Pig
  • Sc-S. cerevisiae
  • Ce-C. elegans
  • Hr-Horse
  • All-All Species Expected

Product Description

This Cell Signaling Technology antibody is conjugated to Alexa Fluor® 488 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.

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.

Protocol

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Flow Cytometry, Methanol Permeabilization Protocol for Directly Conjugated Antibodies

A. Solutions and Reagents

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.

  1. 1X Phosphate Buffered Saline (PBS): To prepare 1 L 1X PBS: add 100 ml 10X PBS (#12528) to 900 ml water mix.
  2. 4% Formaldehyde, Methanol-Free (#47746)
  3. 100% Methanol (#13604): Chill before use
  4. Antibody Dilution Buffer: Purchase ready-to-use Flow Cytometry Antibody Dilution Buffer (#13616), or prepare a 0.5% BSA PBS buffer by dissolving 0.5 g Bovine Serum Albumin (BSA) (#9998) in 100 ml 1X PBS. Store at 4°C.

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/flowdyes for a listing of cellular dyes validated for use in flow cytometry.

B. Fixation

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.

  1. Pellet cells by centrifugation and remove supernatant.
  2. Resuspend cells in approximately 100 µl 4% formaldehyde per 1 million cells. Mix well to dissociate pellet and prevent cross-linking of individual cells.
  3. Fix for 15 min at room temperature (20-25°C).
  4. Wash by centrifugation with excess 1X PBS. Discard supernatant in appropriate waste container. Resuspend cells in 0.5-1 ml 1X PBS. Proceed to Permeabilization step.
    1. Alternatively, cells may be stored overnight at 4°C in 1X PBS.

C. Permeabilization

  1. Permeabilize cells by adding ice-cold 100% methanol slowly to pre-chilled cells, while gently vortexing, to a final concentration of 90% methanol.
  2. Permeabilize for a minimum of 10 min on ice.
  3. Proceed with immunostaining (Section D) or store cells at -20°C in 90% methanol.

D. Immunostaining

NOTE: Count cells using a hemocytometer or alternative method.

  1. Aliquot desired number of cells into tubes or wells. (Generally, 5x105 to 1x106 cells per assay.)
  2. Wash cells by centrifugation in excess 1X PBS to remove methanol. Discard supernatant in appropriate waste container. Repeat if necessary.
  3. Resuspend cells in 100 µl of diluted primary antibody, prepared in Antibody Dilution Buffer at a recommended dilution or as determined via titration.
  4. Incubate for 1 hr at room temperature. Protect from light.
  5. Wash by centrifugation in Antibody Dilution Buffer or 1X PBS. Discard supernatant. Repeat.
  6. Resuspend cells in 200-500 µl of 1X PBS and analyze on flow cytometer.

posted July 2009

revised August 2019

Protocol Id: 407

Specificity / Sensitivity

Galectin-9 (D9R4A) XP® Rabbit mAb (Alexa Fluor® 488 Conjugate) recognizes endogenous levels of total galectin-9 protein.

Species Reactivity:

Human

Source / Purification

Monoclonal antibody is produced by immunizing animals with recombinant human galectin-9 protein.

Background

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).

  1. Barondes, S.H. et al. (1994) Cell 76, 597-8.
  2. Barondes, S.H. et al. (1994) J Biol Chem 269, 20807-10.
  3. Offner, H. et al. (1990) J Neuroimmunol 28, 177-84.
  4. Wells, V. and Mallucci, L. (1991) Cell 64, 91-7.
  5. Filer, A. et al. (2009) Arthritis Rheum 60, 1604-14.
  6. Perillo, N.L. et al. (1995) Nature 378, 736-9.
  7. Cooper, D.N. et al. (1991) J Cell Biol 115, 1437-48.
  8. Gieseke, F. et al. (2013) Eur J Immunol 43, 2741-9.
  9. Zhu, C. et al. (2005) Nat Immunol 6, 1245-52.
  10. Nagahara, K. et al. (2008) J Immunol 181, 7660-9.
  11. Dardalhon, V. et al. (2010) J Immunol 185, 1383-92.
  12. Heusschen, R. et al. (2014) Biochim Biophys Acta 1842, 284-92.
  13. Heusschen, R. et al. (2013) Biol Reprod 88, 22.

Pathways & Proteins

Explore pathways + proteins related to this product.

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.
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 outlicensing@lifetech.com.