Microarrays were prepared at a stiffness of 1 kPa or 25 kPa, as measured via nanoindentation (Optics11 Life Piuma Nanoindenter). Furthermore, an OmniGrid Micro automated microspotter (Digilab, MA) created array grids of 10 ECM proteins in one- and two-way combinations, for a total of 55 combinations (Figure 1A ). The ECM proteins included human collagen I (C1; Millipore, Burlington, MA), collagen III (C3; Millipore), collagen IV (C4; Abcam, Cambridge, MA), collagen V (C5; Abcam), decorin (DC; R&D Systems, Minneapolis, MN), fibronectin (FN, Millipore-Sigma, Burlington, MA), hyaluronic acid (HA; Lifecore Biomedical, Chaska, MN), laminin (LN, Millipore), lumican (LU, Acro Biosystems, Newark, DE), and tenascin C (TC; R&D Systems); these ECM proteins were selected based on our previous work with liver progenitors[19 (link)] and also since these are major ECM proteins present in the liver.[24 (link)] Each microspot or ‘island’ was spotted with a pin diameter of 450 µm and 1 mm center-to-center spacing. Single proteins were spotted at 250 ng µL−1 while 125 ng µL−1 per protein was utilized for two-way combinations. The retention of ECM proteins on PA microarrays was previously characterized using fluorescently labeled C1.[11 (link), 45 ] Lastly, rhodamine-labeled dextran spots were spotted for microarray alignment.
Laminin ln
Manufactured by Merck Group
Sourced in United States
Laminin (LN) is a glycoprotein found in the extracellular matrix of various tissues. It plays a crucial role in cell attachment, migration, and differentiation. Laminin is a key component of the basal lamina, which provides structural support and signaling cues to cells.
Automatically generated - may contain errors
Lab products found in correlation
Poly l ornithine po, by Merck Group (2 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
B27 supplement, by Thermo Fisher Scientific (2 mentions)
Hyaluronic acid, by Lifecore Biomedical (1 mentions)
Influx cell sorter, by BD (1 mentions)
T flasks, by BD (1 mentions)
Epidermal growth factor (egf), by Thermo Fisher Scientific (1 mentions)
Fibroblast growth factor 2 (fgf2), by Thermo Fisher Scientific (1 mentions)
N2 supplement, by Thermo Fisher Scientific (1 mentions)
Insulin, by Merck Group (1 mentions)
Fibronectin fn, by Merck Group (1 mentions)
Bsa bovine serum albumin, by Merck Group (1 mentions)
Collagen type 4, by Merck Group (1 mentions)
5 protocols using laminin ln
1
Extracellular Matrix Protein Microarrays on Polyacrylamide Hydrogels
2
Neural Crest Induction and Migration Assay
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Neural crest induction was performed using monolayer culture protocols as previously described [30 (link)]. In brief, hiPSCs were dissociated into single cells with Accutase and then replated on Matrigel-coated dishes at a density of 20,000 cells/cm2. At the beginning of the induction (day 1), cells were incubated with E6 medium for 24 h and then cultured in NCN2 medium for 6 days (7 days in total). p75highHNK1+ NCSCs were isolated by fluorescence-activated cell sorting (FACS) using a BD Influx Cell Sorter (BD-Pharmingen), cultured and expanded in neural crest culture medium (NCCM).
In the migration assay, day 7 differentiated cells were disaggregated and suspended in neural crest induction medium (NIM) to form spheres in ultralow-attachment culture dishes for 1 day. Then, the spheres were attached to dishes coated with poly-L-ornithine (PO; Sigma-Aldrich) and laminin (LN; Millipore, Temecula, CA, USA) and cultured in NIM for 24–48 h.
In the migration assay, day 7 differentiated cells were disaggregated and suspended in neural crest induction medium (NIM) to form spheres in ultralow-attachment culture dishes for 1 day. Then, the spheres were attached to dishes coated with poly-L-ornithine (PO; Sigma-Aldrich) and laminin (LN; Millipore, Temecula, CA, USA) and cultured in NIM for 24–48 h.
3
Expansion of Human Neural Progenitor Cells
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ReNcell-VM (Millipore®) is a human neural progenitor cell line derived from the ventral mesencephalon region of the fetal brain and immortalized by retroviral transduction with the v-myc oncogene. ReNcell-VM were first expanded in T-flasks (Falcon®, Corning) previously coated with poly-L-ornithine (PO) (20 μg mL−1, Sigma-Aldrich) for 30 min and laminin (LN) (10 μg mL−1 in PBS, Sigma-Aldrich) for 4 h at 37°C and 5% CO2. After seeding, cells were expanded in N2 medium, consisting of DMEM/F12 with glucose (1.6 g L−1, Sigma-Aldrich), N2-supplement (1%, Thermo Fisher), penicillin/streptomycin (1%, Thermo Fisher) and insulin (20 μg mL−1, Sigma-Aldrich), and supplemented with EGF (20 ng mL−1, Peprotech), FGF-2 (20 ng mL−1, Peprotech), and B27 supplement (20 μl mL−1, Thermo Fisher).
4
Extracellular Matrix Protein Preparation
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Fibronectin (Fn) (from bovine plasma), laminin (Ln) (Engelbreth–Holm–Swarm murine sarcoma), collagens I and IV (Cn-I and Cn-IV), mucin (Mu) (type I-S bovine submaxillary glands), and bovine albumin serum (BSA) were purchased from Sigma (St. Louis, MO, USA).
5
Investigating Ovarian Cancer Cell Adhesion
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SKOV3 and OVCAR-3 cells (1.5×104 cells/well) were cultured in triplicate in 24-well plates that had been coated with 1.5% agarose in FBS-free DMEM and RPMI-1640 medium for 10 days, respectively. The cells were exposed to fresh medium every other day and the formation of MCA in individual wells was monitored longitudinally under a microscope. The MCA with a diameter of ≥ 250 μm for SKOV3 or ≥ 50 μm for OVCAR-3 cells was counted because the OVCAR-3 cells grew slowly (Roggiani et al., 2016, Xing et al., 2007).
In addition, the cells were tested in triplicate for the formation of MCA in the presence or absence of different concentrations (10 µg/ml, 20 µg/ml, 40 µg/ml, 80 µg/ml)of FN (RD System), 8 µg/ml Laminin (LN), or 8 µg/ml type IV collagen (Sigma).
Moreover, the cells were tested in triplicate for the formation of MCA in the presence or absence of FN (10 µg/ml for SKOV3, 20 µg/ml for OVCAR-3), together with 5 µg/ml RGD, control RGE (arginine-glycine-glutamic acid (Zhai et al., 2016), Ansp), anti-FN IgG or control IgG (Epitomics).
In addition, the cells were tested in triplicate for the formation of MCA in the presence or absence of different concentrations (10 µg/ml, 20 µg/ml, 40 µg/ml, 80 µg/ml)of FN (RD System), 8 µg/ml Laminin (LN), or 8 µg/ml type IV collagen (Sigma).
Moreover, the cells were tested in triplicate for the formation of MCA in the presence or absence of FN (10 µg/ml for SKOV3, 20 µg/ml for OVCAR-3), together with 5 µg/ml RGD, control RGE (arginine-glycine-glutamic acid (Zhai et al., 2016), Ansp), anti-FN IgG or control IgG (Epitomics).
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