Cell invasion were determined using 24-well microchemotaxis chambers precoated with 7.5 μg collagen type IV (Becton Dickinson Labware), as previously described [57 (link)]. Tumor cells were resuspended in serum-free media and loaded into upper chamber of each well. Cells were incubated at 37°C for 24 h in the presence or the absence of B1f and B3 compounds, after which, the filters were fixed and stained with Diff-Quik (Medion Diagnostic). Cell invasion were quantified by determination of the number of cells that migrated directly through the membrane toward the medium containing 10% serum that was used as a chemoattractant. Cells detected in each well were counted and the results were represented as (number of migrated cells/number of total cells) × 100%.
Collagen type 4
Manufactured by BD
Sourced in United States
Collagen type IV is a structural protein component found in the basement membrane of various tissues, including skin, blood vessels, and kidney glomeruli. It functions as a key structural element, providing support and scaffolding for cells within these tissues.
Automatically generated - may contain errors
Lab products found in correlation
Fibronectin, by BD (6 mentions)
Hepes, by Thermo Fisher Scientific (3 mentions)
Diff quick staining kit, by Siemens (3 mentions)
Hydrocortisone, by Thermo Fisher Scientific (2 mentions)
Bovine platelet poor plasma derived serum, by Thermo Fisher Scientific (2 mentions)
Heparin, by Thermo Fisher Scientific (2 mentions)
Gentamycin, by Thermo Fisher Scientific (2 mentions)
Dmem f12, by Thermo Fisher Scientific (2 mentions)
Knockout serum replacement (ksr), by Thermo Fisher Scientific (2 mentions)
Bone morphogenetic protein 4 (bmp4), by R&D Systems (2 mentions)
Vascular endothelial growth factor (vegf), by R&D Systems (2 mentions)
Basic fibroblast growth factor (bfgf), by Thermo Fisher Scientific (2 mentions)
Biocoat cell culture insert, by BD (2 mentions)
Fibronectin, by Merck Group (2 mentions)
Collagenase type cls 2, by Merck Group (1 mentions)
Hyaluronidase, by Merck Group (1 mentions)
48 well chemotaxis chamber, by Neuro Probe (1 mentions)
Macs magnetic separation column, by Miltenyi Biotec (1 mentions)
Streptavidin microbeads, by Miltenyi Biotec (1 mentions)
Penicillin streptomycin p s, by Thermo Fisher Scientific (1 mentions)
24 protocols using collagen type 4
1
Cell Invasion Assay with Collagen IV
2
Quantification of Cell Migration and Invasion
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Cell migration and invasion were determined using 24-well microchemotaxis chambers alone or precoated with 7.5 μg collagen type IV (Becton Dickinson Labware), respectively [5 (link), 26 (link)]. Control tumor cells or cells expressing Spn4A cDNA were resuspended in serum-free media and loaded into upper chamber of each well. Cells were incubated at 37°C for 24 h, after which, the filters were fixed and stained with Diff-Quik (Medion Diagnostic). Cell migration and invasion were quantified by determination of the number of cells that migrated directly through the membrane toward the medium containing 10% serum that was used as a chemoattractant. Cells detected in each well were counted and the results were represented as (number of migrated cells/number of total cells) × 100%.
3
Isolation and Culture of Rat IMCD Cells
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Inner medullary collecting duct (IMCD) cells were prepared from inner medullas including papilla of sacrificed Wistar rats (license # 84-02.05.20.11.256), as previously described [28 (link)]. Briefly, the inner medullas including papilla of sacrificed Wistar rats were removed, cut into small pieces, and digested in phosphate-buffered saline (PBS) containing 0.2% hyaluronidase (Sigma) and 0.2% collagenase type CLS-II (Sigma) at 37°C for 90 min. Cells were centrifuged at 450 x g, resuspended in PBS and centrifuged once more. Cells were seeded on glass coverslips coated with collagen type IV (Becton-Dickinson) at a density of 105 cells/cm2 and cultured in DMEM containing 100 IU/ml penicillin, 100 μg/ml streptomycin, 0.2% glutamine, 1% nonessential amino acids and 1% ultroser G (BioSepra Inc.) in an 8% CO2 atmosphere.
4
In Vitro Invasion Assay of Tumor Cells
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The in vitro invasion activity of control and MDA-MB-231 ppFurin-expressing cells was performed using 24-well microchemotaxis chambers precoated with 7.5 μg of collagen type IV (Becton Dickinson Labware, Franklin Lake, NJ, USA), as previously described [22 (link),31 (link)]. Tumor cells (1 × 105/mL) were resuspended in serum-free media and were loaded into the upper chamber of Transwells. Cells were incubated at 37 °C for 24 h, and invasion across collagen layer was initiated by adding 10% FBS (chemoattractant) to the lower chamber. Cells detected in each well were counted and the results were represented as (number of migrated cells/number of total cells) × 100%.
5
Cell Invasion Assay with Boyden Chamber
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Cell invasion was evaluated by a modified Boyden chamber method using a 48-well chemotaxis chamber (NeuroProbe, Gaithersburg, MD). Briefly, 25,000 HepG2 and 20,000 Hep3B cells per well were seeded onto an 8 µM pore PDVF membrane (NeuroProbe) previously treated with collagen type IV (BD Biosciences, San Jose, CA) and conditioned medium from scramble- and siPRPF8-treated cells. The lower well was filled with FBS-free medium (negative control) or medium containing 10% FBS, with two replicates per condition23 (link). After 24 h, the non-migrated cells were removed, and the migrated cells were fixed with crystal violet solution (6% glutaraldehyde, 0.5% crystal violet). Images of random fields were acquired and analyzed with ImageJ. The experiments were performed in triplicate.
6
Enrichment and Culture of KDR+ VPC
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KDR+ VPC induced from stage were first labeled with biotinylated anti-VEGF-2/KDR+ ((Miltenyi Biotech) followed by Streptavidin MicroBeads (Miltenyi Biotech), and processed through a MACS magnetic separation column (Miltenyi Biotech) according to the manufacturer recommendations. The KDR+ VPC were plated on dishes coated with either: 10 ng/ml fibronectin (BD Biosciences), 50ng/ml collagen-type IV (BD Biosciences), or 0.5% gelatin at either: 5,000, 10,000, or 20,000 cells/cm2. The stage 2 medium consisted of: 70% alpha-MEM and 30% DMEM, 2Χ Nutridoma CS, 1Χ ps, 1Χ NEAA, 2 mM L-glutamine, and 0.05 mM 2-mercaptoethanol. The supplemental amounts of bFGF examined included: 0 ng/ml, 10 ng/ml, 25 ng/ml, or 50 ng/ml; while amounts of VEGF examined were: 0 ng/ml, 10 ng/ml, 25 ng/ml, or 50 ng/ml. Half media changes were performed every 3 days. The cells were collected 2 weeks following the stage 1 induction, at days 28 and 26 of total differentiation for H7-ESC and H9-ESC, respectively.
7
Shear Stress Differentiation of Pluripotent Cells
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Pluripotent cells were differentiated in medium without LIF and exposed to shear stress, as described previously [13 (link),19 (link)]. As in those studies, glass slides were coated with collagen type IV (3.5 μg/cm2; BD Biosciences, Bedford, MA) for one hour. ESCs and iPSCs were then seeded onto slides at 10,000 cells/cm2 and 40,000 cells/cm2, respectively, to generate similar cell densities just prior to the application of shear. All samples were cultured for two days in differentiation medium (Minimum Essential Alpha Media, 10% fetal bovine serum, 0.1 mM beta-mercaptoethanol, and antibiotics). Fluid flow was applied to cells on slides using a parallel plate bioreactor system, in which medium was recirculated through a flow chamber with a defined channel geometry. Using a calibrated flow rate, a steady laminar shear stress of 5.0 dynes/cm2 was applied for two days (SHEAR). Control samples (STATIC) were cultured under the same time and volume conditions. SHEAR and STATIC samples were imaged and analyzed for gene expression of cytoskeletal proteins.
8
Directed Differentiation of mESCs to Endothelial Cells
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The mESC (A3 and R1 lines) were harvested using trypsin/EDTA (Mediatech) and plated with either 50 ng/mL collagen-type IV (BD Biosciences) or 50 ng/mL fibronectin (BD Biosciences) in basal medium: alpha-minimal essential medium (MEM; Cellgro), 20% KSR (Invitrogen), 1Χ penicillin-streptomycin (ps, Invitrogen), 1Χ nonessential amino acids (NEAA; Invitrogen), 2 mM L-glutamine (Invitrogen), 0.05mM 2-mercaptoethanol (Calbiochem), and 5 ng/mL BMP-4 (R&D Systems), supplemented with either 0 ng/mL, 10 ng/mL, 20 ng/mL, or 30 ng/mL of VEGF (R&D Systems) and induced for between 1–4 days. Experiments were repeated in triplicate (N = 3).
9
In vitro Rat Blood-Brain Barrier Co-Culture
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Primary rat brain microvascular endothelial cells (BMEC) and primary astrocytes necessary for setting up a rat in vitro BBB co-culture model were obtained as reported (Molino et al.65 ,66 (link). Briefly, primary cultures of BMECs, from 5- to 6-week-old Wistar rats, were seeded in the luminal compartment of twelve-well plate polyethylene insert filters (Merck Millipore, Billerica, MA, USA), pre-coated with collagen type IV and fibronectin (BD Biosciences, Franklin Lakes, NJ, USA) to establish the endothelial cell monolayers. Astrocyte primary cultures were prepared from the cerebral cortex of newborn Wistar rats and seeded in the bottom of the twelve-well plates to establish the co-culture with the endothelial cell monolayers in endothelial cell media (ECM) containing DMEM/F12 supplemented with 20% bovine platelet poor plasma derived serum (Alfa Aesar, Ward Hill, MA, USA), basic fibroblast growth factor (bFGF) 2 ng/mL, heparin 100 μg/ mL, gentamycin 50 μg/mL, HEPES 2.5 mM, and hydrocortisone 500 nM (Life Technologies, Carlsbad, CA, USA). Under these conditions, the BMEC monolayers differentiate, express junction-related proteins within 3 days, and remain optimally differentiated during three more days.
10
Breast Cell Morphology and Peroxisome Imaging
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