Bladder cancer cell binding was assessed using 6-well multi-plates, either coated with collagen G type I (400 µg/mL dilution; Sigma-Aldrich, Taufkirchen, Germany), or with human plasma fibronectin (50 µg/mL dilution; BD Biosciences). Uncoated plastic dishes were used to detect unspecific cell binding and served as the controls. The multi-plates were washed twice with BSA (1%). Then, bladder cancer cells (treated vs. non-treated, resistant vs. sensitive) were added at a concentration of 0.5 × 106 cells/well for 60 min at 37 °C. Subsequently, the plates were again washed to remove those cells which did not adhere. Adherent cells establishing firm contact to the well bottom were then fixed with glutaraldehyde (1%; Sigma-Aldrich) and counted microscopically (×200 magnification) in five different fields (0.25 mm2) using a raster ocular. The mean number of adherent cells in the five fields was determined.
Human plasma fibronectin
Manufactured by BD
Sourced in Germany
Human plasma fibronectin is a glycoprotein found in human blood plasma. It plays a core role in cell adhesion, cell motility, and wound healing.
Automatically generated - may contain errors
Lab products found in correlation
Glutaraldehyde, by Merck Group (1 mentions)
Trypsin solution from porcine pancreas, by Merck Group (1 mentions)
Bovine collagen type 1, by BD (1 mentions)
Hepes, by Merck Group (1 mentions)
Sodium bicarbonate, by Merck Group (1 mentions)
Penicillin, by Corning (1 mentions)
Streptomycin, by Corning (1 mentions)
Amphotericin b, by Corning (1 mentions)
Celltiter 96 aqueous one solution cell proliferation assay mts, by Promega (1 mentions)
Fetal bovine serum (fbs), by Innovative Research (1 mentions)
Alexa fluor 647 nhs ester, by Thermo Fisher Scientific (1 mentions)
Biotinylated fibronectin, by Cytoskeleton (1 mentions)
Neutravidin, by Thermo Fisher Scientific (1 mentions)
Sulfo nhs lc biotin, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 555 nhs ester, by Thermo Fisher Scientific (1 mentions)
Sylgard 184, by Dow (1 mentions)
Fluorescent beads, by Thermo Fisher Scientific (1 mentions)
4 protocols using human plasma fibronectin
1
Quantifying Bladder Cancer Cell Adhesion
2
Cell Proliferation Assay with FBS and Reagents
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Fetal bovine serum (FBS) was obtained from Innovative Research (Novi, MI). RPMI-1640 media with L-glutamine and without Sodium bicarbonate was purchased from HyClone Laboratories, Inc. (Logan, UT). CellTiter 96® AQueousOne Solution Cell Proliferation Assay (MTS) was purchased from Promega (Madison, WI). Antibiotic antimycotic solution (100X) stabilized, with 10,000 units penicillin, 10 mg streptomycin and 25 μg amphotericin B per ml (PSA) was obtained from Mediatech, Inc. (Manassas, VA). Human plasma fibronectin and bovine collagen type I was ordered from BD Biosciences (Franklin Lakes, NJ). Sodium bicarbonate, HEPES and trypsin solution from porcine pancreas were purchased from Sigma-Aldrich (St. Louis, MO). T-25 cm2 cell culture flasks were produced by Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).
3
Notch Signaling Cell Culture Protocol
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Human umbilical vein endothelial
cells (HUVEC) and human mesenchymal stem cells (MSC) (Lonza, Walkersville,
MD) were cultured as prescribed by the manufacturer. Chinese hamster
ovary cells harboring Notch “Receiver” and Delta “Sender”
transgenes, [receiver line: CHO-K1-TREx + UAS-H2B-Citrine + CMV-H2B-Cerulean
+ CMV-hNotchECD-Gal4 clone F1; sender line: CHO-K1-TREx + TO-hDll1-mCherry]
both graciously provided by Dr. Michael Elowitz (California Institute
of Technology), were cultured as previously described.34 (link) Human plasma fibronectin (BD Biosciences, Bedford,
MA) was fluorescently labeled using Alexa Fluor 555 NHS ester (Invitrogen,
Carlsbad, CA). Biotinylated fibronectin was obtained from Cytoskeleton,
Inc. (Denver, CO) or made in-house using Biotin-X, SSE, 6-((biotinoyl)amino)hexanoic
acid, sulfosuccinimidyl ester, sodium salt (Sulfo-NHS-LC-Biotin),
(Invitrogen, Carlsbad, CA), and fluorescently labeled using Alexa
Fluor 647 NHS ester (Invitrogen, Carlsbad, CA). Neutravidin and Neutravidin–Oregon
Green 488 conjugate were obtained from Invitrogen. Poly(dimethyl siloxane)
(PDMS; Sylgard 184, Dow Corning, Midland, MI) was used at 10:1 (w:w)
base:curing agent, Young’s modulus ∼1 MPa.
cells (HUVEC) and human mesenchymal stem cells (MSC) (Lonza, Walkersville,
MD) were cultured as prescribed by the manufacturer. Chinese hamster
ovary cells harboring Notch “Receiver” and Delta “Sender”
transgenes, [receiver line: CHO-K1-TREx + UAS-H2B-Citrine + CMV-H2B-Cerulean
+ CMV-hNotchECD-Gal4 clone F1; sender line: CHO-K1-TREx + TO-hDll1-mCherry]
both graciously provided by Dr. Michael Elowitz (California Institute
of Technology), were cultured as previously described.34 (link) Human plasma fibronectin (BD Biosciences, Bedford,
MA) was fluorescently labeled using Alexa Fluor 555 NHS ester (Invitrogen,
Carlsbad, CA). Biotinylated fibronectin was obtained from Cytoskeleton,
Inc. (Denver, CO) or made in-house using Biotin-X, SSE, 6-((biotinoyl)amino)hexanoic
acid, sulfosuccinimidyl ester, sodium salt (Sulfo-NHS-LC-Biotin),
(Invitrogen, Carlsbad, CA), and fluorescently labeled using Alexa
Fluor 647 NHS ester (Invitrogen, Carlsbad, CA). Neutravidin and Neutravidin–Oregon
Green 488 conjugate were obtained from Invitrogen. Poly(dimethyl siloxane)
(PDMS; Sylgard 184, Dow Corning, Midland, MI) was used at 10:1 (w:w)
base:curing agent, Young’s modulus ∼1 MPa.
4
Fabrication of Polyacrylamide Gels
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Polyacrylamide (PA) gels were fabricated following a modified protocol (Pelham & Wang, 1997; Tse & Engler, 2010) (link). Glass slides were activated by incubation in a solution of 0.1 N sodium hydroxide (NaOH), followed by incubation in 2% (v/v) aminopropyl trimethoxysilane (APTMS), then incubation in 0.5% (v/v) glutaraldehyde diluted in phosphate buffered saline (PBS). To fabricate gels with a Young's modulus of 1800 Pa, a solution of 5% acrylamide, 0.06% bisacrylamide, and DI water was degassed for 30 minutes before adding 0.5% (v/v) of ammonium persulphate (10% w/v) and 0.05% (v/v) of tetramethylethylenediamine (TEMED) to initiate polymerization. The solution was then pipetted onto activated glass slides and allowed to polymerize. For traction force microscopy experiments, double-layer PA gels were made by polymerizing a second layer of PA gel containing fluorescent beads (0.2 µm diameter; Life Technologies) on top of the first PA gel layer as described previously (Bridgman, Dave, Asnes, Tullio, & Adelstein, 2001) . Human plasma fibronectin (10 μg/ml; BD Biosciences) was then crosslinked to the PA gel surface with N-Sulfosuccinimidyl-6-(4'-azido-2'nitrophenylamino) hexanoate (sulfo-SANPAH; Pierce) chemistry.
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