Parallel fibronectin stripes 6 µm wide with 10 µm spacing were printed on a glass coverslip using standard microprinting techniques. Briefly, a PDMS stamp was fabricated using soft lithography. Coverslips were cleaned in a boiling mixture solution of ammonium hydroxide and hydrogen peroxide for 10 min, and were rinsed with ethanol and dried under UV light. The coverslips were then treated with plasma for 3 min to increase hydrophilicity. To print fibronectin on the coverslip, fibronectin (50 µg/ml) was applied over the stamp and left for 1 h, and rinsed with PBS. The stamp was then pressed in contact with the coverslip. To reduce non-selective cell attachment, the patterned cover glass was immersed into a blocking reagent (0.2% Pluronics F-127) (Sigma-Alrich) for 1 h. Finally, a ring-shaped thick PDMS wall was bound to the coverslip to form a dish containing a patterned glass bottom. The patterns were examined with NHS-Fluorescein dye (ThermoFisher Scientific) using a fluorescent microscope.
Pluronics f 127
Manufactured by Merck Group
Sourced in United States
Pluronics F-127 is a non-ionic triblock copolymer composed of polyethylene oxide and polypropylene oxide. It is a commonly used surfactant and has applications in various fields, including pharmaceutical, biomedical, and research industries.
Automatically generated - may contain errors
Lab products found in correlation
Sylgard 184, by Dow (4 mentions)
Atto dye, by GE Healthcare (2 mentions)
Mouse c2c12 myoblast, by American Type Culture Collection (2 mentions)
Rhodamine fibronectin, by Cytoskeleton (1 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (1 mentions)
Su 8 photoresist, by MicroChem (1 mentions)
Ar 100, by Thinky (1 mentions)
Alexa 488 c5 maleimide, by Thermo Fisher Scientific (1 mentions)
Hepes, by Thermo Fisher Scientific (1 mentions)
Dylight594 nhs ester, by Thermo Fisher Scientific (1 mentions)
Pf 127, by Merck Group (1 mentions)
Distilled water, by Tedia (1 mentions)
Cremophor el, by Merck Group (1 mentions)
Genistein, by LC Laboratories (1 mentions)
Soluplus, by BASF (1 mentions)
Methanol meoh, by Honeywell (1 mentions)
Fibronectin, by Dow (1 mentions)
Human fibronectin, by BD (1 mentions)
Fibronectin, by Corning (1 mentions)
Up100h, by Hielscher (1 mentions)
17 protocols using pluronics f 127
1
Fabrication of Fibronectin Micropatterns
2
Microposts Functionalization with Fibronectin
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
A flat PDMS stamp was cleaned and then incubated with 50 μg/ml rhodamine fibronectin (Cytoskeleton, Denver, CO, USA) in deionized water for 1 hour. The stamp was dried under sterile airflow and deposited gently on the micropost array, previously subjected to UV ozone treatment. A gentle pressure was applied to the stamp. The contact between the microposts and the stamp was ensured for at least 1 minute. Subsequently the substrates were sterlized in 70% ethanol and immersed in 0.4% Pluronics F127 (Sigma–Aldrich, St. Louis, MO) in PBS for 1 hour to prevent non-specific protein absorption to the non-functionalized surface of the PDMS microposts. Finally the substrates were rinsed with sterile MilliQ water and kept in PBS before use46 (link).
3
Microfluidic Egg Trapping Efficiency
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The outlet of the chip was connected to a syringe pump and a reservoir was attached to the inlet. A syringe pump was used to withdraw the liquid from the outlet. The microchip was prefilled with water before the experiment to minimize bubble formation. The device was loaded on the microscope stage and the sample was applied in the inlet reservoir. To minimize attachment of eggs on the channel surface, 0.1% Pluronics F127 (Sigma-Aldrich, St Louis, MO) was added to the sample. The trapping efficiency was calculated as: captured eggs / (captured eggs + bypassed eggs).
4
Fabrication of Patterned Fibronectin Substrates
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Substrates were fabricated for structural studies as described previously [5 (link), 8 (link), 19 (link), 24 (link), 46 (link)]. Briefly, large cover glass (Brain Research Laboratories, Newton, MA) was cleaned by sonicating, then spin coated with 10:1 Polydimethylsiloxane (PDMS; Ellsworth Adhesives, Germantown, WI). The PDMS coated cover glass was then placed in a 60°C oven to cure overnight (12 h). The cover glass was then cut into smaller individual coverslips to fit in a 12 well plate. Fibronectin (FN; Fischer Scientific Company, Hanover Park, IL) was patterned onto the coverslips in lines 20 μm wide with 5 μm gaps or islands of various aspect ratios using microcontact printing [47 (link)]. The PDMS stamps were then sonicated in ethanol and coated with 0.1 mg/mL drops of FN. After being incubated for 1 h and dried using compressed nitrogen, FN was printed onto the PDMS coated coverslips that were previously exposed to UV light (Jelight Company, Irvine, CA) for 8 min. Finally, the stamped coverslips were submerged in a solution of 5 g Pluronics F-127 (Sigma Aldrich, St. Louis, MO) dissolved in 0.5 L sterile water for 5 min and then rinsed three times with room temperature phosphate-buffered saline (PBS; Life Technologies, Carlsbad, CA). Isotropic tissue samples were made by coating coverslips with a uniform layer of FN [5 (link)].
5
Soft Lithography for Fibronectin Patterning
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Silicon wafers with desired patterns were made using SU-8 photoresist for soft lithography34 . To form stamps, PDMS (Sylgard 184, Dow Corning) at 1:10 mixture ratio (curing agent: silicone elastomer) was molded onto silanized wafers and cured at 80 °C for 2 hr. A mixture of 50 μg/ml pure fibronectin (Roche) and 25 μg/ml conjugated fibronectin (Cy5.5 or Atto dye, GE Healthcare and Sigma) was incubated on the stamps for 1 hr and dried before stamping. Patterns were stamped onto a UV-activated, PDMS spin-coated petri dish and the unstamped area was passivated by 1% Pluronics (F127, Sigma) for 1 hr. Samples were rinsed with PBS before cell seeding.
6
Fabrication of PDMS Microtissue Substrates
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Devices were fabricated as described previously19 (link). Briefly, layers of SU-8 photoresist (Microchem) were patterned onto silicon wafers by successive spin coat, alignment, ultraviolet exposure and baking steps. All masters were developed in a single step in propylene glycol mehyl ether acetate (Sigma) followed by hard bake. Polydimethylsiloxane (PDMS, Sylgard 184, Dow-Corning) microtissue substrates were moulded from the SU-8 masters. Before cell seeding, the PDMS templates were sterilized in 70% ethanol followed by ultraviolet sterilization for 15 min before treatment with 0.02% pluronics-F127 (Sigma) solution for 10 min at room temperature.
7
Fabrication of Adipocyte Patterned Surfaces
8
Quantification of HP1α-Histone Interactions
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Recombinantly expressed and purified HP1α (MW = 22kDa) was purchased (lyophilized against PBS buffer) from Biomatik Corporation (www.biomatik.com ) (Ontario, Canada). Unmodified and methylated histone H3 peptides (1 (link)–22 ) (MW =2354) were purchased in lyophilized form from Bachem Americas,Inc (Torrance, CA) (Bachem.com ) and used as received. Chemicals including Alexa-488 C5-Maleimide, Dylight-594 NHS ester, Nucleic acid dimer sampler kit (YOYO-3 iodide, POPO-1 iodide), dsDNA (NoLimits 2.5kbp, 4kbp, 10kbp) as well as dialysis cassettes (Slide-A-Lyzer G2 3kda cutoff) were obtained from Thermo Fisher Scientific (Waltham, MA). Potassium chloride, Pluronics F127 was obtained from Sigma Aldrich (St. Louis, MO). HEPES ((4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid)) was obtained from Fisher Scientific (Pittsburg, PA). Peptides were reconstituted at 25 mg ml−1 in nuclease-free ultra pure water as per the manufacturer’s instructions and stored as aliquots at −20°.HP1α was reconstituted (0.5 mg ml−1) in nuclease-free ultra pure water as per the manufacturer’s instructions and stored (with 10% glycerol) as flash-frozen 200 ul aliquots in −80°C.
9
Soft Lithography for Fibronectin Patterning
10
Thermosensitive Pluronics F-127 Hydrogel
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!