Cells were kept in suspension or replated on ECM (10 μg/ml, laminin or fibronectin, BD Biosciences)-precoated dishes or coverglasses in the absence (in the case of fibronectin) or presence of low serum (2%, in the case of laminin) for 1 h before being analyzed for cell-ECM adhesion signaling by standard western blotting or for the formation of focal adhesions by indirect immunofluorescence, as described previously [27 (link)]. Function blocking antibodies against human integrin α6 (Millipore, 20 μg/ml) were preincubated with cells by rocking at 37°C for 1 h prior to replating. Pharmacological inhibitors were added to the culture media for 24 h or to replating media at the reseeding time.
Laminin
Manufactured by BD
Sourced in United States, Japan, Germany
Laminin is an extracellular matrix glycoprotein that is a key structural component of the basement membrane. It plays a vital role in cell adhesion, differentiation, migration, and survival. Laminin is a multifunctional protein that supports the attachment, spreading, and differentiation of cells.
Automatically generated - may contain errors
Lab products found in correlation
Poly l ornithine, by Merck Group (13 mentions)
Poly d lysine, by BD (12 mentions)
Fibronectin, by BD (12 mentions)
Poly d lysine, by Merck Group (12 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (9 mentions)
Neurobasal medium, by Thermo Fisher Scientific (8 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (8 mentions)
B27 supplement, by Thermo Fisher Scientific (6 mentions)
Collagen 1, by BD (6 mentions)
Glutamine, by Merck Group (4 mentions)
Penicillin streptomycin, by Merck Group (4 mentions)
Collagenase type 2, by Merck Group (4 mentions)
Poly l lysine (pll), by Merck Group (4 mentions)
Basic fibroblast growth factor (bfgf), by Thermo Fisher Scientific (4 mentions)
Accutase, by Merck Group (3 mentions)
Papain, by Worthington (3 mentions)
Hepes solution, by Merck Group (3 mentions)
Dispase, by Merck Group (3 mentions)
Matrigel, by BD (3 mentions)
Hepes, by Thermo Fisher Scientific (3 mentions)
94 protocols using laminin
1
Cell-ECM Adhesion Signaling Assay
2
Postnatal Mouse Hippocampal Neuron Culture
3
Differentiation of Human ESCs to Hepatocytes
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Human ESCs were passaged with Accutase (Sigma) and plated at a density of 100,000 cells/cm2 in mTeSR with 10 μM Y27632 (Selleck) on Matrigel (BD), Laminin (BD), and collagen IV (BD) (3:1:1) mixed gel coated-plate (Corning). In the restriction of definitive endoderm (DEs) stage (S1), cells were cultured for 24 hrs in RPMI with B27 supplement (1:50, Gibco), 100 ng/ml Activin A (R&D) and 3 μM CHIR99021 (Selleck), and then treated with 100 ng/ml Activin A for 2 days. In the hepatic specification stage to get hepatic progenitor cells (S2), the culture medium was replaced with RPMI (Gibco) supplemented with B27 supplement (1:50), 20 ng/ml BMP4 and 10 ng/ml FGF2 for 5 days. And in the stage of hepatic maturation (S3), cells were cultured in Hepatocyte Culture Medium (HCM, Lonza) with 20 ng/ml HGF, 10 ng/ml OSM and 1 μM dexamethasone for 10–15 days. During stages 2 and 3, cells are fed every 48 hr. The final stage was also carried out with 10 μM SB203580 (Selleck), 50 μM Vitamin K2 (Sigma), 50 μM 2-APB (Tocris), or 0.5 μM Anisomycin (Selleck), and 0.1% dimethyl sulfoxide (DSMO) was used as control, as described in the text. Cells were photographed during differentiation using a Nikon phase contrast microscope (Nikon Microscopes).
4
Induction and Maintenance of EpiLCs from ESCs
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ESCs were cultured in N2B27 basal medium containing 3 μM CHIR99021 (Wako), 0.4 μM PD0325901 (Wako), and LIF in a dish coated with 0.01% poly-L-ornithine (Millipore) and 10 ng/mL laminin (BD Biosciences). Induction of EpiLCs was performed as described previously (Hayashi et al., 2011 (link)). EpiLCs were induced from ESCs in N2B27 basal medium containing 20 ng/mL activin A (Peprotech), 12 ng/mL bFGF (Invitrogen), and 0.1–1% KSR for 2 days on a dish coated with 16.7 μg/mL human plasma fibronectin (Millipore). Two days after EpiLC induction, the cells were collected and replated (0.5–1.0 × 105) with or without doxycycline in GK15 medium on a dish coated with 16.7 μg/mL human plasma fibronectin. For the colony formation assay for AP-positive cells, ESCs, EpiLCs, and EpiLCs induced with or without PRDM14 were dissociated by TrypLE Select (Invitrogen). Cells (1.0 × 104) were cultured under standard ESC culture conditions. After culture for 3 days, the cells were stained for AP activity. The aggregate culture of EpiLCs was performed as previously reported (Nakaki et al., 2013 (link)). In brief, after 36 hr of EpiLC induction the cells were collected, and 2,000 cells per well were transferred to a low-cell binding 96-well plate (NUNC). The cells were grown for 2 days in GK15 medium with or without 1.5 μg/mL doxycycline.
5
Multi-Electrode Array Probe for Axon Elongation
6
Culturing and Maintaining Primary Neuronal Cells
7
Embryonic Neuron Culture with Rotenone Treatment
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Embryonic day 14 (E14) mouse embryos were prepared from a C57BL/6N (Jackson laboratory) pregnant dam as described previously50 (link). The dissected and dissociated tissues were incubated on a 10-mm-diameter ACLAR-embedded file precoated with 50 μg/ml PDL and 4 μg/ml laminin (BD Bioscience, Bedford, MA) in 24-well plates. Culture medium was added the next day and days in vitro at DIV 3. Half of the medium was replaced with an N2-supplemented medium (50% DMEM and 50% Hams F-12) at DIV 5–7. At DIV7, cells were treated with rotenone (1μΜ) alone or in combination with IF1 (10 nM) in N2-supplemented medium (50% DMEM and 50% Hams F-12) for 24 h.
8
Antibody Labeling and Calcium Imaging
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Secondary antibodies were from Cell Signaling Technologies, Fluo-3 AM and Pluronic acid F127 from Invitrogen, United States. Laminin was acquired from BD. All other chemicals and reagents were analytical grade and obtained either from Merck or Sigma-Aldrich.
9
Dissociated Cortical Neuron Culture Protocol
10
Hair Cell Regeneration Assay
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The organ of Corti was plated on a glass coverslip coated in poly-L-ornithine (Sigma) and laminin (BD Biosciences), given 100 μl Dulbecco’s modified Eagle’s medium (DMEM; Invitrogen) with 10% fetal bovine serum (FBS; Invitrogen) and 25 μg/ml ampicillin (Sigma), and cultured at 37°C with 5% CO2. One hour after dissection, cultures were treated with 50 μM gentamicin (Sigma) in DMEM with 10% FBS and ampicillin. The organ of Corti was cultured overnight (16 hr) and then placed in serum-free DMEM:F12 (Invitrogen) with 1% B27 supplement (Invitrogen), 25 μg/ml ampicillin, and either 5 μM dibenzazepine (deshydroxy LY411575; Santa Cruz) or 0.1% DMSO (Sigma) as a control. Undamaged controls were cultured under the same conditions without gentamicin or LY411575. LY411575 treatment continued for 72 hr. After 4 days in vitro, the cultures were fixed with 4% PFA (Electron Microscopy Sciences) and stained for MYO7A to identify hair cells and either DsRed or GFP to enhance the endogenous signal from the reporter.
To measure proliferation, cultures were treated with gentamicin as detailed above followed by 10 μM BrdU along with either LY411575 or 0.1% DMSO for 72 hr and then fixed, treated with 2 N HCl for 30 min at 37°C, and stained for BrdU.
To measure proliferation, cultures were treated with gentamicin as detailed above followed by 10 μM BrdU along with either LY411575 or 0.1% DMSO for 72 hr and then fixed, treated with 2 N HCl for 30 min at 37°C, and stained for BrdU.
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