InnoCyte ECM Cell Adhesion Assay, Galectin-1/Galectin-3 Cat. No. CBA026

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1 User Protocol CBA026 Rev. 12 October 2005 JSW Page 1 of 5 InnoCyte ECM Cell Adhesion Assay, Galectin-1/Galectin-3 Cat. No. CBA026 Table of Contents Page Storage 1 Intended Use 1 Background 1 Principle of the Assay 2 Materials Provided 2 Materials Required but not Provided 2 Reagent Preparation 2 Detailed Protocol 3 Assay Characteristics and Examples 3 References 4 Related Products 4 Storage Upon arrival store the Calcein-AM at -20 C and the remaining components at 4 C. Intended Use The InnoCyte Cell Adhesion Assay, Galectin-1/Galectin-3 is designed for the determination of the relative attachment of adherent cell lines to galectin-1 and galectin-3, for evaluation of cell adhesion receptors, and for screening cell adhesion antagonists. Background The ability of cells to adhere to each other or to constituents of the extracellular matrix (ECM) is important in normal cellular function in mammals. Cell-matrix interactions depend to a large extent upon the engagement of specific ECM proteins with cell surface integrins. Cell adhesion involves protein-protein interaction (recognition of peptide motifs) as well as protein-carbohydrate interaction. For example, selectins are a family of cell-surface adhesion molecules found on leukocytes and endothelial cells that bind to sugar moieties on specific glycoproteins that exhibit mucin-like features. Galectins are a family of carbohydrate binding adhesion molecules (lectins) with affinity for lactose and other β-galactosides. Galectins are characterized by Ca 2+ independence and extensive sequence identity in the carbohydrate recognition domain. Although galectins lack a signal peptide and are normally found in the cytosol, they can be externalized by an atypical secretory mechanism. Galectins are known to modulate cell adhesion, cell growth, cell transformation, embryogenesis, and apoptosis. Galectin-8 is reported to be as effective as fibronectin in promoting cell adhesion via ligation of cell surface integrin receptors. Galectin-1 is a monomeric or homodimeric prototype galectin that is expressed in a variety of cells. Galectin-1 is involved in multiple biological functions, including cell adhesion, apoptosis, and tumor metastasis. Galectin-1 can modulate cell-cell as well as cell-matrix interactions. Galectin-3 is expressed in normal and neoplastic cells and regulates cell growth, cell adhesion, differentiation, and cell death. There is evidence to suggest that galectin-3 plays an important role in tumor progression and metastasis. Recombinant galectin-3 is reported to bind α1β1 integrin, a receptor for laminin and collagen. Galectin-3 is also a substrate for MMP-2 and MMP-9 cleavage, resulting in a 22 kda fragment containing a carbohydrate recognition domain and 9 kda fragment containing the N- terminal part of galectin-3.

2 User Protocol CBA026 Rev. 12 October 2005 JSW Page 2 of 5 In conclusion, Galectin-1 and galectin-3 interactions with galactoside-bearing ligands seem to be important in many processes such as fetal development, tissue remodeling, apoptosis, and tumor cell adhesion and metastasis. Due to their strong implication in inflammation and cancer they may be useful targets for development of new antiinflammatory and anti-cancer therapies. Principle of the Assay The InnoCyte Cell Adhesion Assay, Galectin-1/Galectin-3 is designed for the analysis of relative cell attachment to ECM proteins. The kit is supplied with a 96-well strip plate coated with galectin-1 and galectin-3 (see plate format below). Cells are seeded in the coated wells and incubated at +37 C. Following incubation, the wells are washed briefly and attached cells are labeled the green fluorescent dye, calcein-am. BSA-coated wells serve as a negative control and poly-l-lysine-coated wells serve as a positive control for general attachment. Relative cell attachment is assessed using a fluorescence plate reader. Materials Provided Galectin-1/Galectin-3-Coated 96-Well Plate (Kit Component No. JA7990): packaged as a single 96-well plate, including frame, pre-coated as follows: Galectin-1-Coated 96-well Plate Strips: Three 2 x 8-well strips coated with recombinant human galectin- 1, BSA, or poly-l-lysine (see configuration below) Galectin-3-Coated 96-well Plate Strips: Three 2 x 8-well strips coated with recombinant human galectin- 3, BSA, or poly-l-lysine (see configuration below) Row A is coated with BSA, rows B G are coated with galectin-1 (Gal-1) or galectin-3 (Gal-3), and row H is coated with poly-l-lysine (P-Lys) A BSA BSA BSA BSA BSA BSA BSA BSA BSA BSA BSA BSA B Gal-1 Gal-1 Gal-1 Gal-1 Gal-1 Gal-1 Gal-3 Gal-3 Gal-3 Gal-3 Gal-3 Gal-3 C Gal-1 Gal-1 Gal-1 Gal-1 Gal-1 Gal-1 Gal-3 Gal-3 Gal-3 Gal-3 Gal-3 Gal-3 D Gal-1 Gal-1 Gal-1 Gal-1 Gal-1 Gal-1 Gal-3 Gal-3 Gal-3 Gal-3 Gal-3 Gal-3 E Gal-1 Gal-1 Gal-1 Gal-1 Gal-1 Gal-1 Gal-3 Gal-3 Gal-3 Gal-3 Gal-3 Gal-3 F Gal-1 Gal-1 Gal-1 Gal-1 Gal-1 Gal-1 Gal-3 Gal-3 Gal-3 Gal-3 Gal-3 Gal-3 G Gal-1 Gal-1 Gal-1 Gal-1 Gal-1 Gal-1 Gal-3 Gal-3 Gal-3 Gal-3 Gal-3 Gal-3 H P-Lys P-Lys P-Lys P-Lys P-Lys P-Lys P-Lys P-Lys P-Lys P-Lys P-Lys P-Lys Calcein-AM Solution (Kit Component No. JA7705): 50 µl in an amber vial D-PBS (Kit Component No. JA7706): 10 ml in a plastic bottle Materials Required but not Provided Cells and cell culture medium Fluorescence reader capable of measuring fluorescence in 96-well plates at an excitation wavelength or ~485 nm and an emission wavelength of ~520 nm 1X PBS for plate washing Preparation of Reagents Calcein-AM Working Solution: prepare immediately prior to use; the Calcein-AM Working Solution cannot be stored for future assays. Warm the Calcein-AM Solution and the D-PBS to room temperature Dilute an appropriate amount of Calcein-AM Solution 1:300 with D-PBS For example: to prepare 6 ml Calcein-AM Working Solution add 20 µl Calcein-AM Solution to 5980 µl D-PBS.

3 User Protocol CBA026 Rev. 12 October 2005 JSW Page 3 of 5 Detailed Protocol 1. Grow cells of choice in culture medium as appropriate. 2. Harvest the cells by centrifugation at 1000 rpm for 5 min at room temperature. Adherent cells may be trypsinized, followed by centrifugation at 1000 rpm for 5 min at room temperature. Resuspend the cell pellet in serum-free culture medium. The recommended cell density is 100,000 to 500,000 cells/ml. 3. Remove required number of strips from the Galectin-1/Galectin-3-Coated 96-Well Plate and place them in the 96-well frame. Return the unused strips to the foil pouch and reseal the entire edge. Store unused strips at 4 º C. 4. Add 100 µl of the cell suspension from step 2 (10,000-50,000 per well), in duplicate, to the desired wells of the Galectin-1/Galectin-3-Coated 96-Well Plate and incubate for 1-2 h at 37 C in a cell culture incubator. 5. Discard the cell suspension by shaking the contents of the wells into an approved biological waste container. Gently wash the plate by adding 200 µl 1X PBS to each well; discard the wash by shaking the contents of the wells into an approved biological waste container. Repeat the wash. 6. Add 100 µl Calcein-AM Working Solution to each well. 7. Incubate for 1 h at 37 C in a cell culture incubator. 8. Measure the fluorescence in each well using a fluorescence plate reader at an excitation wavelength of ~485 nm and an emission wavelength ~520 nm. Assay Characteristics and Examples RFU (485/520 nm) Galectin-1 Galectin-3 BSA A431 SK-BR-3 SW-480 THP-1 HT-1080 HeLa Figure 1: Relative cell attachment of various cell lines to galectin-1, galectin-3, and BSA. Approximately 40,000 cells were added to wells coated with galectin-1, galectin-3, and BSA and incubated for 1.5 h at 37 C in the presence of 6% CO 2. Cells were washed gently with D-PBS and labeled with Calcein-AM for 1 h at 37 C in the presence of 6% CO 2. Fluorescence was measured as indicated in the Detailed Protocol. HT-1080 cells displayed appreciable binding to poly-l-lysine, which served as a positive control (data not shown).

4 User Protocol CBA026 Rev. 12 October 2005 JSW Page 4 of RFU (485/520 nm) mm lactose 2.5 mm lactose 10 mm lactose Galectin-1 BSA Figure 2: Inhibition of HT-1080 cell attachment to galectin-1. Approximately 40,000 cells were added to wells coated with galectin-1 in the presence of 0 mm, 2.5 mm, or 10 mm lactose and incubated for 1.5 h at 37 C in a cell culture incubator in the presence of 6% CO 2. Cells were washed gently with D-PBS and labeled with calcein-am for 1 h at 37 C in a cell culture incubator in the presence of 6% CO 2. Cell attachment to galectin-3 is also inhibited by lactose (data not shown). References Takenaka, Y., et al Glycoconjugate Journal 19, 543. Horiguchi, N., et al J. Biochem. 134, 689. Levy, Y., et al J. Biol. Chem. 276, Hynes, R.O., et al Proc. Natl. Acad. Sci. USA 96, Kaltner, H., et al Acta Anatomica 161, 162. Perillo, N. L., et al J. Mol. Med. 76, 402. Warfield, P Biochem. Biophys. Res. Commun 246, 788. Akahani, S., et al Cancer Res. 57, Horowitz, A.F., et al Trends Cell Biol. 6, 460. Barondes, S.H., et al J. Biol. Chem. 269, Ochieng, J., et al Biochemistry 33, Related Products InnoCyte ECM Cell Adhesion Assay, Fibronectin (Cat. No. CBA011) InnoCyte ECM Cell Adhesion Assay, Vitronectin (Cat. No. CBA012) InnoCyte ECM Cell Adhesion Assay, Collagen Type IV (Cat. No. CBA013) InnoCyte ECM Cell Adhesion Assay, Laminin/Basement Membrane Complex (Cat. No. CBA025) InnoCyte α 2,α 3,α 5 Integrin Detection Kit (Cat. No. CBA037) Lactose

5 User Protocol CBA026 Rev. 12 October 2005 JSW Page 5 of 5 Trademarks Calbiochem is a registered trademark of EMD Biosciences, Inc. InnoCyte is a trademark of EMD Biosciences, Inc. Prices and availability are subject to change. Copyright 2005 EMD Biosciences, Inc., an affiliate of Merck KGaA, Darmstadt,. All rights reserved. Each product is sold with a limited warranty which is provided with each purchase. Each product is intended to be used for research purposes only. It is not to be used for drug or diagnostic purposes nor is it intended for human use. EMD Biosciences products may not be resold, modified for resale, or used to manufacture commercial products without written approval of EMD Biosciences.