Maxinunc microtiter plates (eBiosciences) were coated with 0.5 μg human collagen I (Corning), human collagen VI (Abcam), bovine fibronectin (Corning), mouse collagen IV (Corning) and mouse laminin (Corning) at 4°C for overnight, and blocked with 3% BSA for 1 h at 37 °C. Recombinant His-tagged BBA33 protein was serially diluted 1:2 starting from 4 μM down to 62.5 nM in PBS, 0.1% Tween-20, and added to coated wells in triplicate and incubated for 1 h at 37 °C. After five washes in PBS, 0.1% Tween-20, a 1:6000 dilution of monoclonal antibody directed against poly-His (His6) was added to each well and incubated for 1 h at 37°C. Following five washes in PBS, 0.1% Tween-20, a 1:4000 dilution of HRP conjugated antimouse IgG was added to each well and incubated for 1 h at 37°C. The wells were then washed in PBS, 0.1% Tween-20, after which 3,3′,5,5′-tetramethylbenzidine was added as substrate. The enzymatic reaction was stopped after 3 min using 0.16 M sulfuric acid (Thermo scientific), and the absorbance at 450 nm was determined.
Mouse laminin
Manufactured by Corning
Sourced in United States
Mouse laminin is a key component of the extracellular matrix. It is a large glycoprotein that provides structural support and promotes cell adhesion and differentiation in various cell types.
Automatically generated - may contain errors
Lab products found in correlation
Neurobasal medium, by Thermo Fisher Scientific (2 mentions)
Glutamax, by Thermo Fisher Scientific (2 mentions)
Bovine fibronectin, by Corning (1 mentions)
Sulfuric acid, by Thermo Fisher Scientific (1 mentions)
Mouse collagen 4, by Corning (1 mentions)
Bml gr100, by Enzo Life Sciences (1 mentions)
Dmem f12, by Thermo Fisher Scientific (1 mentions)
Poly l ornithine, by Merck Group (1 mentions)
Poly l lysine (pll), by Merck Group (1 mentions)
Alexa fluor 488 phalloidin, by Thermo Fisher Scientific (1 mentions)
Bz x800 microscope, by Keyence (1 mentions)
Bgj398, by Selleck Chemicals (1 mentions)
Heparin sodium salt, by Merck Group (1 mentions)
Human fibronectin, by Corning (1 mentions)
Mouse collagen type 4, by Corning (1 mentions)
Rat tail collagen type 1, by Corning (1 mentions)
6 protocols using mouse laminin
1
Quantification of BBA33 Protein Binding
2
Hippocampal Neural Stem Cell Culture and Differentiation
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Hippocampal neural stem (HCN) cells isolated from adult rats were cultured at 37 °C and 5% CO2 on dishes coated with 10 μg/mL poly-l -ornithine (Sigma-Aldrich, P3655) and 5 μg/mL mouse laminin (Corning, 354232) and maintained in Dulbecco’s modified Eagle’s medium (DMEM)/F-12 (Invitrogen, 12400-024) containing 5 mg/L insulin (Sigma-Aldrich, I6634), 100 mg/L apo-transferrin (Prospec, PRO-325), penicillin (100 U/L)/streptomycin (100 mg/L) (HyClone, SV30010), 16 mg/L putrescin (Sigma-Aldrich, P5780), 30 nM sodium selenite (Sigma-Aldrich, S1382), 20 nM progesterone (Sigma-Aldrich, P0130), and 20 ng/ml basic fibroblast growth factor (bFGF) (Peprotech, #100-18B)20 (link). For differentiation, HCN cells were plated into coated dishes at a density of ~0.5 × 105 cells/cm2, and 24 h later, the culture medium was replaced with medium containing 1 µM retinoic acid (Enzo, BML-GR100), 5 µM forskolin (Enzo, BML-CN100), and 0.1% fetal bovine serum (Tissue Culture Biologicals, 101)21 (link). Half of the medium was replaced every 2 days until the experiment.
3
Growth Cone Collapse Assay for Sema3A
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A primary culture of mouse DRG neurons was prepared as previously described (Kawashima et al., 2021 (link)). Briefly, DRGs were dissected from embryonic day 14–15 WT (C57B6/J) and Crmp1ki/ki mice and plated on glass-bottom culture dishes precoated sequentially with poly-L-lysine (100 μg/ml; #P4832; Sigma-Aldrich) and mouse laminin (8 μg/ml; #354232; Corning). DRG explants were subsequently cultured in 250 μl/well of Neurobasal medium (#21103049; Thermo Fisher Scientific) supplemented with 2% B-27 (#17504044; Thermo Fisher Scientific), 1 mm GlutaMax (#35050061; Thermo Fisher Scientific), 20 ng/ml NGF, 50 U/ml penicillin, and 50 μg/ml streptomycin for 1 d at 37°C. The explants were stimulated with either 0.5, 1, or 3 U/ml purified recombinant chick Sema3A for 10 min at 37°C and fixed with PBS containing 2% formaldehyde and 10% sucrose. Growth cones were stained with Alexa Fluor 488 phalloidin (#A12379; Thermo Fisher Scientific). The growth cones without lamellipodia or filopodia were scored as collapsed. In each condition, >50 growth cones were examined. Sema3A at 1 U/ml concentration induced 50% collapsed growth cones of chick E7 DRG neurons (Nakamura et al., 2014 (link)). Growth cone images were captured with a BZ-X800 microscope at 40× magnification (Keyence).
4
ARPE-19 Cells for FGF Receptor Modulation
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ARPE-19 cells stably expressing S179C-TIMP3, wild-type-TIMP3 (WT), or vector alone were reported previously [19 (link)]. Cells were expanded in DMEM-F12 with 10% FBS before transfer to polyester inserts coated with mouse laminin (Corning Inc., Corning, NY, USA, #23017). 720,000 cells and 100,000 cells were plated per well in each well of a 12-well plate or 24-well plate, respectively using a previously published protocol [36 (link)]. Essentially, ARPE-19 cells were cultured for at least 2 weeks in nicotinamide-supplemented media with 1% FBS. Media were replaced twice per week. Cells were serum-starved for 24 h before treatment with the FGF Receptor inhibitor BGJ-398 (Selleckchem, Houston, TX, USA, #S2183) for 48 h. Similarly, cells were treated with FGF-2 (Gibco from Thermo Fisher Scientific, #13256-029) with the required cofactor heparin sodium salt (1 μg/mL, Sigma Aldrich, #H3149) for 48 h after serum starving for 24 h.
5
Nanofiber Scaffold Co-Culture for Neuromuscular Junction
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A 15 cm × 15 cm PCL aligned nanofiber sheet was custom fabricated and purchased from Nanofiber Solutions LLC (Ohio, USA). The sheets were cut into 10mmx5mm pieces, placed in 24 well tissue culture plates and UV sterilized prior to coating with 20 µg/mL poly-D-lysine (PDL) in sterile cell culture water overnight. The sheets were subsequently washed thrice with PBS before coating with 20 µg/mL mouse laminin (Corning,354232) for 2 h. Pre-differentiated C2C12 cells were plated on the nanofiber sheets at a concentration of 2 × 105 cells/sheet in growth media for 24 h before being cultured using differentiation media for 7 days in vitro (DIV) with regular changes of media. Dissociated motor neurons were plated on top of the myocyte layer at a concentration of 1 × 105cells/sheet and the coculture was maintained with serum-free motor neuron media up to 14DIV with regular changes of media. The sheets with only myocytes were also kept on serum-free motor neuron media between 7-14DIV to maintain parity of cell culture condition between groups.
6
Extracellular Matrix Components for Cell Culture
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Rat tail collagen type I (#354236), mouse laminin (#354232), mouse collagen type IV (#354233), and human fibronectin (#356008) were from Corning.
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