Poly l lysine (pll)

Manufactured by Merck Group

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Poly-L-lysine is a synthetic polymer composed of the amino acid L-lysine. It is commonly used as a coating agent for various laboratory applications, such as cell culture and microscopy. Poly-L-lysine enhances the attachment and growth of cells on surfaces by providing a positively charged substrate.

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3 215 protocols using poly l lysine (pll)

Coating Polystyrene Microcarriers with PLL and LN

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Polystyrene MCs with an average diameter of 97 ± 10 μm were purchased from Thermo-Fisher Scientific (Waltham, MA, USA). These MC beads were sterilised by exposure to gamma irradiation (10 minutes, 10 kGray/hour) in a ⁶⁰Co irradiator (Gammacell 200 Excel; Ottawa, ON, Canada) before being utilised for culture.
For coating with PLL (70 to 150 kDa; Sigma-Aldrich, St. Louis, MO, USA), 12 μl of 1 mg/ml PLL were added to 20 mg MC in 600 μl phosphate-buffered saline (PBS), to make a final PLL concentration of 20 μg/ml. This was incubated at 4°C overnight and then rinsed with PBS before further coating. Similarly, the coating of murine LN (Life Technologies, Carlsbad, CA, USA) was achieved by adding 20 μl of 1 mg/ml LN to 20 mg PLL-coated MC in 600 μl PBS, to make a final LN concentration of 33 μg/ml [35 (link)]. The MCs were similarly incubated at 4°C overnight, followed by a rinse with PBS before being used for cell culture.

Lam A.T., Chen A.K., Li J., Birch W.R., Reuveny S, & Oh S.K. (2014). Conjoint propagation and differentiation of human embryonic stem cells to cardiomyocytes in a defined microcarrier spinner culture. Stem Cell Research & Therapy, 5(5), 110.

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Fabrication of Multilayer (PLL/HA)-(PSS/PAH) Films

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PLL (poly-L-lysine, MW = 5.7 × 10⁴ Da, Sigma, St. Quentin Fallavier, France) and HA (hyaluronic acid, MW = 4.0 × 10⁵ Da, BioIberica, Barcelona) were to construct the (PLL/HA)₂₄ films, and PSS (poly sodium 4-styrenesulfonate, MW = 7.0 × 10⁴ Da, Sigma, St. Quentin Fallavier) and PAH (poly allylamine hydrochloride, MW = 7.0 × 10⁴ Da, Sigma) for the (PSS/PAH) capping films, which were deposited on top of the (PLL/HA)₂₄ strata. PLL, HA, PSS, and PAH were dissolved at 1 mg/mL in a buffer solution containing 150 mM NaCl and 20 mM of tris(hydroxymethyl)-aminomethan (TRIS, Merck) at pH 7.4, and all rinsing steps were performed in the same buffer. (PLL/HA)₂₄ strata and (PSS/PAH) capping films were prepared using a dipping machine (Dipping Robot DR3, Riegler & Kirstein GmbH, Berlin, Germany), on glass slides (VWR Scientific, Fontenay sous Bois, France). The apparent modulus of the (PLL/HA)₂₄-(PSS/PAH) film was approximately 20 kPa^{41 (link),42 (link)}. For this film, we shall used the short-hand notation E₂₀ (Fig. S5A,B).

Rabineau M., Flick F., Ehlinger C., Mathieu E., Duluc I., Jung M., Senger B., Kocgozlu L., Schaaf P., Lavalle P., Freund J.N., Haikel Y, & Vautier D. (2018). Chromatin de-condensation by switching substrate elasticity. Scientific Reports, 8, 12655.

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Surface Preparation for Cell Culture

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After oxygen plasma treatment the carbon and glass surfaces were sterilized by rinsing with 96% ethanol three times and then completely dried. One set of all carbon and glass substrates was then coated with 20μg/ml poly-L-lysine (PLL, 1g/mL; Sigma, St Louis, MO, USA) in PBS and the TCP control with 10μg/ml PLL in PBS for 4 hours prior to cell seeding. Another set of all substrates used for cell culture experiments directly without any bio-coating, was treated similarly except for the PLL coating step. All samples (controls, glass and carbon surfaces) were prepared in parallel for all experimental conditions i.e., scanning electron microscopy and immuno-cytochemistry.

Asif A., García-López S., Heiskanen A., Martínez-Serrano A., Keller S.S., Pereira M.P, & Emnéus J. (2020). Pyrolytic Carbon Nanograss Enhances Neurogenesis and Dopaminergic Differentiation of Human Midbrain Neural Stem Cells. Advanced healthcare materials, 9(20).

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Hippocampal Cell Culture Protocol

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Four hippocampi taken from two E17 ICR mice were digested with 0.25% papain containing 1% DNase in Glucose mix at 37°C for 5 min. Dissociated cells were seeded on PEI-coated 10-cm culture dishes in Dulbecco's Modified Eagle Medium (DMEM)/Ham’s F12 medium (Sigma-Aldrich) containing 5% horse serum (GIBCO) and 5% fetal bovine serum (JRH Biosciences, Lenexa, KS, USA), 1% L-glutamine, 5 U/mL penicillin and 5 μg/mL streptomycin. Medium was changed every three days. After confluency, which took 7–10 days, cells were suspended with 0.05% trypsin in Ca and Mg free Hank’s balanced salt solution (Nacalai tesque) and plated on poly-L-Lysine (PLL, Sigma-Aldrich) coated round glass coverslips (5 × 10⁴ cells/cover slip) or PLL coated 6-well plate (3 × 10⁵ cells/well) with the same culture medium. Within 2–5 days after the re-plating when cultures reached 70–90% confluency, cells were used for experiments.

Adachi C., Otsuka S, & Inoue T. (2022). Cholesterol-induced robust Ca oscillation in astrocytes required for survival and lipid droplet formation in high-cholesterol condition. iScience, 25(10), 105138.

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Encapsulation of Raw 264.7 Cells in APA Microcapsules

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Raw 264.7 cells were encapsulated into non-homogeneous alginate-poly-L-lysine-alginate (APA) microcapsules prepared using an electrostatic droplet generator (Nisco Engineering AG, Zurich, Switzerland). Briefly, cells were harvested from monolayer cultures using a cell scraper and suspended at a density of 4 × 10⁶ cells/ml in 1.5% (w/v) low-viscosity high gluluronic acid alginate (LVG) (NovaMatrix/FMC Biopolymer Corporation, Oslo, Norway) dissolved in a 1% mannitol solution and previously filtered through a 0.20 µm syringe filter (Millipore, Madrid, Spain). This suspension was extruded through a sterile 0.17 mm inner diameter needle using a 10 ml sterile syringe with a peristaltic pump into a calcium chloride solution (55 mM). The resulting alginate beads were maintained in agitation for 10 min in this solution. Then, the microcapsules were successively chemically cross-linked with poly-L-lysine 0.05% (w/v) for 5 min (PLL; MW 15.000–30.000 Da; Sigma-Aldrich, St. Louis, MO) and then coated with LVG-alginate 0.1% (w/v) for 5 min. Microcapsules were prepared at room temperature and under sterile conditions. Subsequently, they were maintained under normal Raw 264.7 culture conditions.

Sola A., Saenz del Burgo L., Ciriza J., Hernandez R.M., Orive G., Martin Cordero J., Calle P., Pedraz J.L, & Hotter G. (2017). Microencapsulated macrophages releases conditioned medium able to prevent epithelial to mesenchymal transition. Drug Delivery, 25(1), 91-101.

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Magnetized Nanoparticle Cellular Uptake

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The magnetic IONPs of γ‐Fe₂O₃@PSC were synthesized according to the previous protocols.31 (link) To promote cellular uptake of IONPs, poly-L-lysine (PLL) was used to modify the γ‐Fe₂O₃@PSC by mixing 10 g/mL PLL (Sigma-Aldrich, St. Louis, MO, USA) with 5 mg/mL γ‐Fe₂O₃@PSC and sonicating for at least 3 h.
The morphology and hydrodynamic diameter of γ‐Fe₂O₃@PSC and γ‐Fe₂O₃@PSC /PLL were characterized by TEM (FEI Tecnai G2 F30, Hillsboro, USA) and dynamic light scattering (DLS) using a ζ-potential laser particle size analyzer (Litesizer 500, Anton Paar, Graz, Austria), respectively.

Han X., Tang S., Wang L., Xu X., Yan R., Yan S., Guo Z., Hu K., Yu T., Li M., Li Y., Zhang F, & Gu N. (2021). Multicellular Spheroids Formation on Hydrogel Enhances Osteogenic/Odontogenic Differentiation of Dental Pulp Stem Cells Under Magnetic Nanoparticles Induction. International Journal of Nanomedicine, 16, 5101-5115.

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Micropatterned PDMS-Gel Hybrid Chambers

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PDMS solution was spin-coated onto a silicon wafer at 1,600 rpm for 60s. The PDMS thin film was partially cured for 30 min at 70°C. 1 mm × 1 mm culture microwells were cut in a gel film (PF Gel-Film, gel thickness 150 μm, Gel-Pack, Hayward, CA, USA) with a vinyl plotter (Graphtec Robo CE-4000, Graphtec America Inc., Irvine, CA, USA). The square microwells were then pressed on top of the PDMS film. An additional curing phase of at least 2 h at 70°C was applied to irreversibly bond the assembly of the two levels of PDMS and gel film. The chambers were sterilized with deep UV treatment (185 and 254 nm, 13 mW/cm2, 5 min). The chambers were then washed two times with ethanol, one time with sterilized deionized water. Cell culture chambers were coated with 100 μg/ml PLL (Sigma-Aldrich, St. Louis, MO, USA), rinsed three times with sterilized deionized water and air-dried during at least 2 h. Cells were either added at this step (PLL-coated coverslips) or after an additional overnight coating with 10 μg/ml laminin (Sigma-Aldrich, St. Louis, MO, USA) at 37°C.

Dupin I., Lokmane L., Dahan M., Garel S, & Studer V. (2015). Subrepellent doses of Slit1 promote Netrin-1 chemotactic responses in subsets of axons. Neural Development, 10, 5.

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Biochip Surface Functionalization for Neuron Culture

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The surface of a newly fabricated PDMS biochip is hydrophobic and thus not suitable for cell attachment and growth. Therefore, the biochip was treated for 10 min with oxygen plasma, which replaces the -CH3 groups with -OH groups, converts the surface from hydrophobic to hydrophilic, and adds some -O⁻ groups, which give the surface a negative charge. The cell membrane is also negatively charged. To reverse the biochip's charge and thereby improve neuronal attachment and growth, it was immersed in positively charged 0.01 mg/ml poly-L-lysine (PLL, Sigma) diluted in Milli-Q filtered water in a 35 mm dish at room temperature overnight, which provided enough time to ensure that the microunit could physically absorb PLL molecules. The biochip was rinsed three times in 48 hr in MIlli-Q water. This procedure was repeated to produce enough biochips to perform the experiments.
Following PLL-coating, the biochips, except for those to be used as control groups, were immersed in 25 μg/ml recombinant human NCAM-L1/Fc chimera (L1, R&D systems) diluted in Milli-Q water for 4 hr at 37°C and rinsed once in M199 medium before neuronal culture.

Wei L., Sweeney A.J., Sheng L., Fang Y., Kindy M.S., Xi T, & Gao B.Z. (2014). Single-Neuron Axonal Pathfinding under Geometric Guidance: Low-Dose-Methylmercury Developmental Neurotoxicity Test. Lab on a chip, 14(18), 3564-3571.

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Preparation and Characterization of PLGA-PLL Nanoparticles

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To prepare the PLGA nanoparticles coated with PLL (Sigma-Aldrich), preformed PLGA nanoparticles (10 mg/ml) in ultrapure water were added dropwise into 1 ml of 0.05% (w/v) PLL under 700 rpm of stirring for 2 hours. Next, five washes were used to remove any free polyelectrolyte, and the PLLNP were resuspended in ultrapure water for further use. To characterize the size and zeta potential of the PLGA cores and PLLNP, the samples were tested using a Zetasizer MAL 1267090 (Malvern Panalytical).

Zhang F., Li Z., Duan Y., Luan H., Yin L., Guo Z., Chen C., Xu M., Gao W., Fang R.H., Zhang L, & Wang J. (2022). Extremophile-based biohybrid micromotors for biomedical operations in harsh acidic environments. Science Advances, 8(51), eade6455.

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Scaffold-Mediated Lentiviral Transduction of MSCs

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Scaffold disks were incubated with 0.002% PLL (Sigma-Aldrich) overnight to facilitate non-covalent association of viral particles with the scaffold surface through charge interaction between the positively charged PLL and negatively charged LV[37 , 41 (link), 42 (link)]. Scaffolds were then rinsed in PBS (Gibco) and incubated with concentrated LV for 1.5 hours, rinsed again, and seeded with 465K MSCs (16.7e6 cells/mL) at the end of passage 4 [37 ]. The biological titer of concentrated LV for all scaffold-mediated transduction experiments was measured as 1.6 × 10⁷ transducing units/mL. MSCs were also seeded directly onto rinsed, PLL-coated scaffolds to produce non-transduced (NT) MSC constructs. One hour after seeding, expansion medium was added to each sample.

Glass K.A., Link J.M., Brunger J.M., Moutos F.T., Gersbach C.A, & Guilak F. (2014). Tissue-engineered cartilage with inducible and tunable immunomodulatory properties. Biomaterials, 35(22), 5921-5931.

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