Laminin 511

Manufactured by BioLamina

Sourced in Sweden

Laminin-511 is a component of the extracellular matrix (ECM) that provides structural support and regulates cellular behavior. It is a large protein complex composed of five different subunits, forming a sheet-like structure. Laminin-511 plays a crucial role in the attachment, migration, and differentiation of various cell types.

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Laminin (9 citations) Human laminin-511 (3 citations) Recombinant human laminin-511 (3 citations) Recombinant laminin-511 (2 citations)

12 protocols using laminin 511

Culturing and Transfecting Cell Lines

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R1/E mESCs were cultured in DMEM (high glucose, pyruvate; Gibco) supplemented with 16% FBS (Gibco), antibiotic–antimycotic (Invitrogen), nonessential amino acids (Gibco), β-mercaptoethanol (Gibco), and recombinant mouse leukemia inhibitory factor (ESGRO). For routine culturing, cells were passaged every 48 h and seeded at a density of 35,000 cells/cm² onto gelatin-coated dishes. HeLa Kyoto, HEK293, and Ptk₂ cell lines were cultured in DMEM (high glucose, pyruvate) supplemented with 10% FBS and antibiotic–antimycotic (Invitrogen) and passaged routinely. Prior to imaging, cells were seeded onto wells of 4-well imaging dishes (Ibidi) or 24-well imaging dishes (Ibidi). To support growth in adherent monolayer, mESCs were seeded onto wells coated with 5 µg/ml laminin-511 (BioLamina) in 1× PBS (supplemented with Ca²⁺ and Mg²⁺). All cells were maintained at 37°C and 5% CO₂.
Transfection of HeLa Kyoto cells was performed using Lipofectamine 3000 (Thermo Fisher Scientific) according to the manufacturer’s instructions.

Kletter T., Reusch S., Cavazza T., Dempewolf N., Tischer C, & Reber S. (2021). Volumetric morphometry reveals spindle width as the best predictor of mammalian spindle scaling. The Journal of Cell Biology, 221(1), e202106170.

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Culturing hiPS Cells on ECM Coatings

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Tissue culture-treated plates were incubated for 2 h at room temperature with 5 μg/mL of collagen I (BD Biosciences), laminin 511 (BioLamina), laminin 511-E8 (Iwai North America), or laminin 521 (BioLamina). hiPS cells were dissociated by treatment with enzyme-free cell dissociation buffer (GIBCO) and cultured on the ECM-coated surfaces at 4×10⁴ cells/cm² in serum-free DMEM/F12 (GIBCO) for 3 h. For prolonged culture, the cells were fed daily with mTeSR1 medium.

Musah S., Mammoto A., Ferrante T.C., Jeanty S.S., Hirano-Kobayashi M., Mammoto T., Roberts K., Chung S., Novak R., Ingram M., Fatanat-Didar T., Koshy S., Weaver J.C., Church G.M, & Ingber D.E. (2017). Mature induced-pluripotent-stem-cell-derived human podocytes reconstitute kidney glomerular-capillary-wall function on a chip. Nature biomedical engineering, 1, 0069.

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Wnt Signaling Activation and Imaging

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Tissue culture grade glass bottom 24-well plates (MatTek) were treated with laminin-511 (20 μg ml⁻¹) (Biolamina) for 4 h at 37 °C and plated with cells at approximately 2,500 cells per cm². Cells were exposed to Wnt3a (50–100 ng ml⁻¹) and Shield1 (50–100 nM) at the time of plating. After approximately 16 h, cells were selected for time-lapse imaging based on system activation, assessed by visible mTurquoise2 signal, and then imaged in an incubated microscope environment every 14 min over 20–40 h before being immediately fixed. Samples were fixed with 4% formaldehyde in PBS for 5 min. Samples cultured for smFISH imaging, but without time-lapse video tracking, were prepared similarly (typically with a higher plated cell density) and activated for different lengths of time, as stated.

Frieda K.L., Linton J.M., Hormoz S., Choi J., Chow K.H., Singer Z.S., Budde M.W., Elowitz M.B, & Cai L. (2016). Synthetic recording and in situ readout of lineage information in single cells. Nature, 541(7635), 107-111.

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Isolation and Expansion of Human Corneal Endothelial Progenitors

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HCEPs were isolated and cultured as previously described by Hara et al. [24 (link)]. Briefly, Descemet’s membranes with HCECs were stripped from the human corneas using sterile surgical forceps. The tissue was transferred to an enzyme cell detachment medium (Accutase, Life Technologies) at 37°C for 30 minutes, and centrifuged at 15,000 rpm for 5 minutes. The cells were seeded at a density of 100 to 300 cells/cm² onto culture plates coated with 20 μg/mL laminin-511 (BioLamina).
The culture medium comprised Dulbecco’s modified Eagle’s medium/nutrient mixture F-12 (DMEM/F12), supplemented with 20% knockout serum replacement (KSR), 2mM L-glutamine, 1% nonessential amino acids, 100μM 2-mercaptoethanol, 50 U/mL penicillin G, and 50 μg/mL streptomycin (all from Life Technologies) along with 4 ng/mL basic fibroblast growth factor (bFGF) and 10μM Y-27632 (both from Wako Pure Chemical Industrials). Paired corneas from two donors were used to compare the expansion of HCEPs with and without 10μM Y-27632. HCEPs were cultured in a humidified atmosphere with 5% CO2 at 37°C, and the culture medium was changed every 2 to 3 days. When the cells reached confluence, they were harvested with Accutase and passaged at ratios of 1:2 to 1:4.

Shin H., Min J.K., Kim N.R., Seo K.Y., Chin H.S., Lee S, & Jung J.W. (2022). Effects of Y-27632, a Rho-associated Kinase Inhibitor, on Human Corneal Endothelial Cells Cultured by Isolating Human Corneal Endothelial Progenitor Cells. Korean Journal of Ophthalmology : KJO, 37(1), 31-41.

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Modulation of T cell activation and polarization by laminins

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CD4 T cells from C57BL/6 mice were activated in 96-well plates at a density of 1 × 10⁵ in complete RPMI medium. Cells were activated with coated anti-CD3 (1 or 5 μg/mL, clone 2C11, BD Bioscience) with or without coated laminin 411(2 μg/mL, Biolamina, Matawan, NJ) and/or laminin 511 (1 μg/mL, Biolamina). After 3 days of culture, T cell proliferation and CD69, CD25 and CD44 expression were determined by flow cytometry. For T cell polarization, CD4 T cells from C57BL/6 mice were cultured 5 days with coated anti-CD3 mAb (5μg/mL) and soluble anti-CD28 (1μg/mL, clone 37.51, eBioscience, San Diego, CA) with or without coated laminin 411 and /or laminin 511, in pro-Th1, Th2, Treg or Th17 environments. The laminin α5 receptor was blocked with anti- α6 integrin mAb or with anti- α dystroglycan mAb. All the antibodies were preservative and endotoxin free. After 5 days of culture or after CD4 T cell isolation from the spleen or the LNs, CD4 T cells were activated for 4 hours with phorbol 12-myristate13-acetate (PMA) (40 ng/mL, Sigma-Aldrich, Allentown, PA) and ionomycin (1μg/mL, Sigma-Aldrich) in the presence of brefeldin A (dilution 1:1000, BD Biosciences) and cytokine expression was analyzed by flow cytometry.

Simon T., Li L., Wagner C., Zhang T., Saxena V., Brinkman C.C., Tostanoski L.H., Ostrand-Rosenberg S., Jewell C., Shea-Donohue T., Hippen K., Blazar B., Abdi R, & Bromberg J.S. (2019). Differential Regulation of T Cell Immunity and Tolerance by Stromal Laminin Expressed in the Lymph Node. Transplantation, 103(10), 2075-2089.

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Monitoring Pluripotency Dynamics in ESCs

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Mouse ESC lines were cultured in NACL or 2i/LIF medium on Laminin 511 (BioLamina)-coated 8-well slides (Ibidi) and imaged at 20 min intervals for 6 days in mCherry and Venus fluorescent light channels, in 5% CO₂ and 20% O₂ at 37°C under a Deltavision Widefield Screening microscope. ESCs were seeded at 300 cells/cm² 48 hr before the beginning of the time lapse for NACL and PD03 experiments, or at 30,000 cells/cm² 24 hr before for PrE differentiation. For PD03 experiments, cells were cultured in NACL for at least two passages and PD03 was added just before starting the time lapse. For PrE experiments, cells were cultured in 2i/LIF for 2 passages and then changed to RPMI minimal medium just before starting the experiment. Activin A, Chiron, and LIF (RACL) were added 24 hr after the time lapse started. In all experiments, the media was changed every day of the time lapse. To test that the laminin coating was not affecting the behaviour of the HFHCV cell line, we sorted Hhex-high and low populations seeded in both gelatine and laminin and analysed them 24 and 48 hr later to test that their re-equilibration rates are the same.

Perera M., Nissen S.B., Proks M., Pozzi S., Monteiro R.S., Trusina A, & Brickman J.M. (2022). Transcriptional heterogeneity and cell cycle regulation as central determinants of Primitive Endoderm priming. eLife, 11, e78967.

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Culturing hiPS Cells on ECM Coatings

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Ghost RBCs Phagocytosis Assay

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To induce formation of ghost RBCs, 0.5 × 10⁶ RBCs were incubated with magnetic cell sorting–enriched trophozoites overnight at 37°C on laminin-511–coated (0.5 μg; BioLamina, Sundyberg, Sweden) microscopic chambers (Nunc Laboratory-Tek Chambered Coverglass; ThermoFisher Scientific). uRBCs were labeled with Celltrace carboxyfluorescein diacetate succinimidyl ester (ThermoFisher Scientific), and iRBCs were stained with 1 μg/mL Hoechst (Sigma, Spruce). Monocytes were isolated from peripheral blood mononuclear cells (PBMCs) from healthy donors by density gradient centrifugation with Percoll (Pharmacia). Monocytes were isolated from PBMCs by using CD14 MicroBeads (Miltenyi Biotec, Bergisch Gladbach, Germany) magnetic cell sorting labeling technology. CD14⁺ monocytes and 10% active normal human serum (AB+, pooled from 5 healthy donors) was then added to the overnight-incubated RBCs. Phagocytosis was visualized with Observer Z1 (Zeiss) taking frames every 0.5 minutes of a defined area on the microscopic chamber under a ×40 oil objective for 1 hour.

Dalimot J.J., Klei T.R., Beuger B.M., Dikmen Z., Bouwman S.A., Mombo-Ngoma G., Zoleko-Manego R., Ndzebe-Ndoumba W.F., Egée S., Kuijpers T.W., Grobusch M.P, & van Bruggen R. (2022). Malaria-associated adhesion molecule activation facilitates the destruction of uninfected red blood cells. Blood Advances, 6(21), 5798-5810.

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Laminin-511 Coating for Cell Culture

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Glass coverslips were coated using 5 μg/μl laminin-511 (Biolamina, Sweden) and incubated at 4°C for 16 h.

Cottle L., Gan W.J., Gilroy I., Samra J.S., Gill A.J., Loudovaris T., Thomas H.E., Hawthorne W.J., Kebede M.A, & Thorn P. (2021). Structural and functional polarisation of human pancreatic beta cells in islets from organ donors with and without type 2 diabetes. Diabetologia, 64(3), 618-629.

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Extracellular Matrix Replacement for hEPS Cells

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To screen extracellular matrix that can functionally replace Matrigel, selected matrix proteins were coated according to manufacturers’ suggestions. The coated plates were used immediately or sealed and stored at 4 °C for no >2 weeks. All the experiments were performed at least in 3 replications for each matrix protein, and at least two EPS cell lines were used to confirm the experimental results. hEPS cells were harvested at 1.5, 24, and 72 h after seeding, and the cell number was counted using hemocytometer. Violet staining was also performed to further confirm the results. The matrix proteins tested were as follows: Matrigel (Corning, 54248), Geltrex (Thermo Fisher Scientific, A1413202), Fibronectin (Sigma, F2006), Laminin (Sigma, L6274), Collagen (Sigma, C5533G), VTN-N (Thermo Fisher Scientific, A14700), Laminin 511 (Biolamina, LN511-0202) and Laminin 521 (Stem cell technologies, 77004).

Liu B., Chen S., Xu Y., Lyu Y., Wang J., Du Y., Sun Y., Liu H., Zhou H., Lai W., Xue A., Yin M., Li C., Bai Y., Xu J, & Deng H. (2021). Chemically defined and xeno-free culture condition for human extended pluripotent stem cells. Nature Communications, 12, 3017.

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