Cell adhesion assays were performed using 96-well round-bottomed microtiter plates (Immulon-2HB; Dynex Technologies, Inc.). The wells were coated overnight at 4°C with 10 μg/ml human fibronectin (GIBCO BRL), collagen I (Nitta Gelatin Co., Japan), laminin 111 (laminin 1; Invitrogen), laminin 211 (laminin 2; Invitrogen), and human laminin 511/521 (laminin10/11; R&D Systems), and then diluted with PBS and blocked with 3% bovine serum albumin for 1 hour at 37°C. After washing, 10,000 cells were added and incubated for 60 minutes at 37°C. Antibodies against β1, α6, and α4 integrins, or the RGD and RAD peptides were added to the culture medium as potential inhibitors of cell adhesion. The plates were washed 3 times with PBS to remove unattached cells, and then the attached cells were identified by staining with crystal violet and evaluated using spectrophotometric analysis (wavelength; 600 nm).
Laminin 211
Manufactured by Thermo Fisher Scientific
Sourced in Japan, Niger
Laminin 211 is a protein found in the extracellular matrix. It serves as a structural component and provides a scaffold for cell attachment and organization.
Automatically generated - may contain errors
Lab products found in correlation
Immulon 2hb, by Dynex (1 mentions)
Laminin 111, by Thermo Fisher Scientific (1 mentions)
Human fibronectin, by Thermo Fisher Scientific (1 mentions)
Collagen 1, by Nitta Gelatin (1 mentions)
P gsk3β, by Cell Signaling Technology (1 mentions)
Gapdh, by LI COR (1 mentions)
Pakt473, by Cell Signaling Technology (1 mentions)
Odyssey image analyzer, by LI COR (1 mentions)
Antibody dilution buffer, by LI COR (1 mentions)
Protease and phosphatase inhibitor, by Roche (1 mentions)
Nitrocellulose membrane, by LI COR (1 mentions)
Gapdh, by Cell Signaling Technology (1 mentions)
Empiria studio software, by LI COR (1 mentions)
2 protocols using laminin 211
1
Cell Adhesion Assay Protocols
2
Western Blot Analysis of Ventricular Proteins
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For Western blotting of total proteins, left ventricular tissues from KO and Control mice were homogenized in a modified RIPA buffer containing protease and phosphatase inhibitors (Roche, CA). Samples were run on an SDS-PAGE gel and transferred to a nitrocellulose membrane (Li-Cor Biosciences, NE) using protocols published by our laboratory. Incubation with primary antibodies for PGM1, (HPA024637, Sigma-Aldrich), Laminin-211 (PA1-16730, Invitrogen), Dystroglycan (11017-AP Proteintech), pAkt473 (#9271,Cell signaling Technology), pGsk3β (#9336,Cell signaling Technology), Gapdh (#14C10,Cell signaling Technology) was conducted at a dilution of 1:1000 in antibody dilution buffer (Li-Cor Biosciences, NE) as described previously. 16 Primary antibodies were detected with IR dye-conjugated secondary antibodies and visualized by Odyssey Image Analyzer (Li-Cor Biosciences, NE). Quantitative analysis of the fluorescence signals was performed by Empiria Studio software (Li-Cor Biosciences, NE), and the results were normalized to the corresponding Gapdh abundance detected in the same samples.
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