Laminin 111

Manufactured by Merck Group

Sourced in United States

Laminin-111 is a protein found in the extracellular matrix. It is a key structural component of basement membranes and plays a role in cell adhesion and differentiation.

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Murine laminin-111 Mouse laminin 111 Anti-Laminin-111 Laminin

7 protocols using laminin 111

Preparation and Characterization of Laminin Polymers

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The aLam polymers were made by diluting laminin-111 (L2020 1 mg/ml; Sigma Aldrich, St. Louis, MO, USA) in filtered and autoclaved 20 mM sodium acetate buffer (pH 4) in a sterile hood to a final working concentration of 100 μg/ml (Fig. 1A). It was shown that in this buffer, laminin forms polymers with a regular hexagonal configuration (Fig. 1A) by linking the short arms of the heterotrimer [4 (link)]. The polymerization state of the polymer was confirmed with dynamic light scattering [9 ] and scanning electron microscopy (Fig. 1B, C). Previously, we [5 ] and others [4 (link)] have demonstrated that once polymers are formed at low pH they retain their structure [4 (link)] and growth-promoting ability [5 ] when introduced into a neutral pH environment.

Haggerty A.E., Bening M.R., Pherribo G., Dauer E.A, & Oudega M. (2019). Laminin polymer treatment accelerates repair of the crushed peripheral nerve in adult rats. Acta biomaterialia, 86, 185-193.

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Hydrogel Scaffold Fabrication with DTPA

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3,3 Dithiopropionic acid (DTPA), anhydrous methanol, anhydrous ethanol, hydrazine hydrate (HH), hexane, concentrated sulfuric acid, ethyl ether, hydrochloric acid (HCl), sodium hydroxide (NaOH), sodium chloride (NaCl), hyaluronic acid sodium salt (HA) from Streptococcus equi, N-3-dimethylaminopropyl-N′-ethylcarbodiimide hydrochloride (EDC), 5,5'-dithiobis-2-nitrobenzoic acid (Ellman’s reagent), and laminin-111 were purchased from Sigma Aldrich (St. Louis, MO, USA). Dithiothreitol (DTT) was purchased from Gold Biotechnology (St. Louis, MO, USA). Poly(ethylene glycol) divinyl sulfone (PEGDVS) 5 kDa was purchased from JenKem Technology USA (Allen, TX, USA).

Addington C., Heffernan J., Millar-Haskell C., Tucker E., Sirianni R, & Stabenfeldt S. (2015). Enhancing neural stem cell response to SDF-1α gradients through hyaluronic acid-laminin hydrogels. Biomaterials, 72, 11-19.

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ECM and Synthemax Coating Protocols

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TCPS plates were coated with 0.61 μg of ECM protein per cm² ECM (fibronectin (Sigma, St. Louis, MO), laminin 1,1,1 (Sigma), or collagen type 1 (Nutragen, Advanced Biomatrix, San Diego, CA)) in phosphate buffered saline (PBS) and placed in an incubator overnight at 37°C. For Synthemax plates, TCPS plates were coated with 3.3 μg per cm² Synthemax (Corning) in PBS and incubated overnight at 37°C. Prior to cell seeding, plates were washed with sterile PBS to remove any non-adsorbed coating.

Laperle A., Masters K.S, & Palecek S.P. (2014). Influence of substrate composition on human embryonic stem cell differentiation and extracellular matrix production in embryoid bodies. Biotechnology progress, 31(1), 212-219.

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Quantifying Fibroblast Adhesion and Migration

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Cell adhesion was measured as described^{23 (link)} with slight modifications. Culture plates were coated with bovine serum albumin (BSA) (1 mg/mL), laminin 111 (Sigma-Aldrich, 10 μg/mL) and collagen I (Millipore, 10 μg/mL), according to the manufacturer’s instruction. About 0.5×10⁶ fibroblasts (subject and control) were seeded per well using serum-free media (OptiPRO, Invitrogen), and allowed to attach for 90 min at 37°C with 5% CO₂. Non-adherent cells were washed off with PBS and the plate was frozen for 30 min. After thawing, cells were lysed with CyQuant dye (Invitrogen) diluted in PBS for 10 min; the fluorescence was read at 520 nm (excitation 480 nm, emission 520 nm). All experiments were performed in triplicate; three independent assays were performed.
For migration assays, fibroblasts were grown to 80% confluence in non-coated chamber slides. At confluence, the medium was changed to serum-free, and a 0.9 mm scratch was made in the confluent layer of cells. Cells were labelled with Calcein AM (Invitrogen), and the migration of the cells into the wound area was imaged at 12, 24, 36, 48 and 60 h using a fluorescent microscope. All experiments were performed in triplicate; six independent assays were performed.

Vilboux T., Malicdan M.C., Chang Y.M., Guo J., Zerfas P.M., Stephen J., Cullinane A.R., Bryant J., Fischer R., Brooks B.P., Zein W.M., Wiggs E.A., Zalewski C.K., Poretti A., Bryan M.M., Vemulapalli M., Mullikin J.C., Kirby M., Anderson S.M., Huizing M., Toro C., Gahl W.A, & Gunay-Aygun M. (2016). Cystic cerebellar dysplasia and biallelic LAMA1 mutations: a lamininopathy associated with tics, obsessive compulsive traits and myopia due to cell adhesion and migration defects. Journal of medical genetics, 53(5), 318-329.

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Cell Adhesion Assay on ECM-Coated Plates

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First, 48-well dishes were coated with 0.1% gelatin (G1393, Sigma Aldrich), 10 µg/mL collagen type I from rat tail (C7661, Sigma Aldrich), 10 µg/mL collagen type II from chicken sternal cartilage (C9301, Sigma Aldrich), and 1 mg/mL laminin-111 (L2020, Sigma Aldrich) solution and blocked with 0.25% BSA (Sigma Aldrich) in PBS. BSA-coated wells were used as a negative and non-specific control. The wells were rinsed with PBS before the experiment. Cells were suspended in PBS (+Ca/Mg, Gibco) and incubated with antibodies for 30 min before plating the cells. The isotype control IgG2a antibody (401504, Biolegend) and blocking monoclonal integrin α10 antibody were used at a concentration of 3‒5 µg/mL. The C2C12α10 and C2C12α11 cells were added to the wells at a concentration of 65,000/well and U3054MG cells at a concentration of 100,000/well, and cells were allowed to adhere for 1 h at 37 °C. After incubation, cells were gently rinsed with PBS (+Ca/Mg) to remove non-adherent cells. Adherent cells were fixed with ethanol, stained with 0.09% crystal violet (Sigma Aldrich), and the absorbed dye was extracted by 10% acetic acid (Sigma Aldrich). The extraction was quantified by measuring the absorbance at 560 nm in the SpectraMax i3 multi-mode plate reader (Molecular Devices).

Munksgaard Thorén M., Chmielarska Masoumi K., Krona C., Huang X., Kundu S., Schmidt L., Forsberg-Nilsson K., Floyd Keep M., Englund E., Nelander S., Holmqvist B, & Lundgren-Åkerlund E. (2019). Integrin α10, a Novel Therapeutic Target in Glioblastoma, Regulates Cell Migration, Proliferation, and Survival. Cancers, 11(4), 587.

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Membrane Staining of C2C12 Myoblasts

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C2C12 cells (American Type Culture Collection, CRL-1772; RRID: CVCL-0188) were cultured as described previously (12 (link)). For microscopy, myoblasts were plated on glass-bottom dishes (IBIDI, 81158) coated with laminin 111 (Sigma-Aldrich, L2020). Four days after differentiation induction, cells were stained with CellMask Green Plasma Membrane Stain (5 μg/ml; Thermo Fisher Scientific, C37608) for 7 min and imaged live on a Zeiss Cell Observer SD confocal microscope (Carl Zeiss) using a 63× oil objective [numerical aperture (NA) 1.40] and a QImaging ROLERA EM-C2 electron-multiplying charge-coupled device (EMCCD) camera. CellMask Green Plasma Membrane Stain was excited with a 488-nm laser line, and the respective emission was collected with an FE01-520/35 emission filter.

Kwan H.L., Chan Z.C., Bi X., Kutkowska J., Prószyński T.J., Chan C.B, & Lee C.W. (2023). Nerve-independent formation of membrane infoldings at topologically complex postsynaptic apparatus by caveolin-3. Science Advances, 9(24), eadg0183.

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hNPC Culture and PAMAM Exposure

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HNPCs derived from H9 human embryonic stem cells were obtained from Chemicon-Millipore (ENStem Human Neural Progenitor Expansion Kit, Norcross, GA, USA). hNPCs were cultured in neural expansion medium (NEM; ENStem-A) with 0.05% b-FGF and 1% L-glutamine (Sigma-Aldrich) on poly-ornithine-coated (Sigma-Aldrich) and laminin-111-coated (Sigma-Aldrich) dishes prior to PAMAM exposure for cell maintenance. During the PAMAM exposure, the cells were cultured in NDM, which consisted of a mixture of neurobasal medium with 1  B27, 1  N2 and 10 ng/mL of brainderived neurotrophic factor (Invitrogen, Carlsbad, CA, USA), and 2 mM glutaMAX (Gibco, Life Technologies, Carlsbad, CA, USA) on 0.54 mg/cm 2 laminin-511-coated (BioLaminaAB, Stockholm, Sweden) 96-well plates. Cells were seeded in 96-well microplates at a density of 1 × 10 5 cells/well in 200 μL of NDM; three parallel sets of plates were used. After 24 hr of cell attachment, the culture medium was replaced with the test solution or a control solution (NDM with 0.1% methanol or isopropanol).

Zeng Y., Kurokawa Y., Win-Shwe T.T., Zeng Q., Hirano S., Zhang Z, & Sone H. (2016). Effects of PAMAM dendrimers with various surface functional groups and multiple generations on cytotoxicity and neuronal differentiation using human neural progenitor cells. The Journal of toxicological sciences, 41(3).

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