Giant polymersome (GUV) formation was accomplished according to the standard electroformation technique [10] using a Nanion Ve sicle Prep Pro setup (Nanion Technologies, Munich, Germany). A freshly cleaned ITO-coated glass slide was first plasma-treated (Plasma Cleaner, PDC-002, Harrick Plasma, Ithaca, New Yo rk, USA), and then, a thin polymer film was deposited on it. For control GUVs (only PMOXA-b-PDMS-b-PMOXA) a solution of PMOXA-b-PDMS-b-PMOXA in ethanol (6 mg/mL, 40 µL) was dispersed on the ITO-coated side of the glass slide and ethanol was subsequently evaporated using a vacuum chamber for 30 min (Plasma Cleaner, PDC-002, Harrick Plasma, Ithaca, New Yo rk, USA). For the heparin GUVs a mixture of PMOXA-b-PDMS-ing polymersomes were formed at a larger size (micrometer scale, heparin GUVs) to study malaria protein and whole parasite interaction with RBC-sized polymersomebased mimics (Fig. 1). The same two block copolymers previously used for nanomimic formation were also used to form these giant mimics. PDMS 65 -b-heparin 12 was mixed with PMOXA 5 -b-PDMS 58b-PMOXA 5 (or PMOXA 9 -b-PDMS 67 -b-PMOXA 9 ) using 15 wt% of the first block copolymer and subsequent electroformation, yielding heparin GUVs. GUVs made from PMOXA 5 -b-PDMS 58 -b-PMOXA 5 without the heparin-functionalized copolymer were used as controls.
Plasma cleaner pdc 002
Manufactured by Harrick
Sourced in United States
The Plasma Cleaner PDC-002 is a compact, benchtop instrument designed for cleaning and activating surfaces. It utilizes a low-pressure plasma discharge to remove organic contaminants and enhance surface wettability. The device features a user-friendly interface and is suitable for a variety of applications.
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Lab products found in correlation
Btpe 50, by Instech (1 mentions)
Milli q, by Merck Group (1 mentions)
Micromanipulator, by Eppendorf (1 mentions)
Inverted spinning disc confocal microscope, by Oxford Instruments (1 mentions)
P8920, by Merck Group (1 mentions)
Hepes, by Merck Group (1 mentions)
Tecnai g2 spirit twin transmission electron microscope, by Thermo Fisher Scientific (1 mentions)
Cover glasses, by Thermo Fisher Scientific (1 mentions)
Jfc 1600 auto fine coater, by JEOL (1 mentions)
Eclipse ci s, by Nikon (1 mentions)
Fitc bsa, by Thermo Fisher Scientific (1 mentions)
Nanion vesicle prep pro setup, by Nanion Technologies (1 mentions)
Su 8 2010, by MicroChem (1 mentions)
15 protocols using plasma cleaner pdc 002
1
Giant Heparin-Functionalized Polymersomes
2
Microfluidic Mother Machine for Individual Cell Observation
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Microfluidic mother machines allow straightforward, stable culture and observation of individual cells for hundreds of generations, in contrast to microscope culture techniques which do not actively remove progeny during growth. Our devices (23 µm × 1.3 µm × 1.3 µm (l, w, h) growth channels with 5 µm spacing along a split media trench, Supplementary Fig. 2 ) are fabricated by curing degassed polydimethylsiloxane (Sylgard 184, 1:10 catalyst:resin) against epoxy replicate master molds produced from primary wafer-molded devices54 ,55 (link). Cured PDMS bulk is peeled from the molds and trimmed as appropriate, input and output ports are punched with electropolished 18ga luer stubs. The PDMS bulk and a clean cover slip are rinsed with 100% isopropanol, blown dry, baked on a hotplate at ~125 °C for 15 min. They are then cooled, and the surfaces to be bonded exposed to air plasma (Harrick PDC-002 plasma cleaner, medium power) for 1 min, and then brought gently into contact. The bonded devices are left at room temperature for 15 min, post baked for 1–2 h at 80 °C, and then stored until use. Polyethylene tubing (Instech, BTPE-50) is press-fitted onto 22ga luer stubs and cannulae (Instech) on opposite ends for coupling to media supplies and waste, and to the devices, respectively.
3
Characterization of Polymer Nanocomposite Surfaces
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Example 1
Materials.
Poly(2-vinyl pyridine-b-dimethylsiloxane) block copolymer P5321 (P2VP-b-PDMS 16,000-b-10,000 g/mol) was purchased from Polymer Source, Inc., Canada. 7 nm silica nanoparticles, 1,2-bis(triethoxysilyl)ethane, (3-bromopropyl)trimethoxysilane, dichloromethane, 1,2-dichloroethane and anhydrous toluene were all purchased from Sigma-Aldrich and used as received. Non-woven clothlike wipes made from cellulose and polypropylene blends were used as received from workwipes. Water purified in a Milli-Q (Millipore) system was used during all the experiments.
Characterization.
Air plasma treatment were carried out using PDC-002 plasma cleaner (Harrick Plasma company, US). Scanning electron microscopy (SEM) images were obtained on FEI Magellan scanning electron microscope. Contact angle measurements were performed with an Attension Theta system (KSV Instruments Ltd., Finland) at ambient temperature. Water droplets of 2 μL were used for the water contact angles measurement in air. For the underwater oil contact angles measurements, oil droplets (1,2-dichloroethane, ca. 2 μL) were dropped carefully onto the surface of the samples, which were fixed on the bottom of a glass container filled with water of different pHs. An average CA value was obtained by measuring the same sample at three different positions.
4
Establishing Inert Cell Substrate
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Glass coverslips with ridges were rinsed with isopropanol, ethanol and dH2O, air-dried and plasma cleaned (pdc-002 plasma cleaner, Harrick). They were then incubated with Poly-2-methyl-2-oxazoline (1 mg/ml for 1h at RT; PAcrAm™-g-(PMOXA); SuSoS Surface Technology) or PLL-PEG (1 mg/ml for 1h at RT; PLL-PEG; SuSoS Surface Technology) to generate an inert, non-adhesive coating. Agarose blocks (1%) were generated as described above and matured DCs or purified naïve t-cells were injected under the agarose using a micropipette. Spinning-disc confocal microscopy and TIRF microscopy were performed as described above.
5
Investigating Cell Membrane Mechanics
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Glass bottom dishes (50 mm dish diameter, 14 mm glass diameter, glass coverslips No. 1, Mattek) were plasma cleaned (pdc-002 plasma cleaner, Harrick) and coated with 1x poly-L-lysine (P8920, Merck) in dH2O for 10 min. Dishes were washed twice with dH2O and then dried for at least 4h at room temperature. Cells in R10 (mDCs or t-cells expressing LifeAct-eGFP) were incubated for 15 min at 37 °C and dishes were carefully washed once with R10 containing HEPES (10mM; Sigma) to remove floating cells. Dishes were immediately mounted on an inverted spinning-disc confocal microscope (Andor) equipped with a micromanipulator (Eppendorf) and maintained at 37°C in a custom-built climate chamber. Micropipettes (blunt; inner diameter 4 μm; bent angle 30°) (BioMedical Instruments) were centrally positioned over the cell and carefully lowered to indent the cell body. Movies were recorded using a 100x/1.4 NA objective and a 488 nm laser line. Z-stacks of 3 image (0.5 μm step size) were recorded every two seconds.
6
TEM Imaging of Polymer Samples
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For transmission electron
microscopy (TEM) experiments, the polymers were dissolved in DI water
to a final concentration of 20 g L–1 and stored
at room temperature. Four hundred mesh copper–rhodium grids
(maxtaform) with a homemade carbon layer were glow discharged in air
for 1.5 min at medium power in a Harrick PDC-002 plasma cleaner. The
20 g L–1 sample was diluted (1/125 or 1/625), and
8 μL was incubated on the grids for 1 min before blotting (Whatman
filter paper No. 50). The grids were washed with water (three times)
and 2% w/v uranyl acetate (three times). After the last dose of uranyl
acetate was applied, the grid was left to incubate for 5 min before
blotting. A single-tilt room temperature holder in an FEI Tecnai G2
Spirit TWIN transmission electron microscope equipped with a tungsten
emitter at 120 kV was used. Images were recorded with an Eagle CCD
camera under low-dose conditions. The micrographs were binned two
times resulting in a pixel size of 2.2 Å per pixel at specimen
level.
microscopy (TEM) experiments, the polymers were dissolved in DI water
to a final concentration of 20 g L–1 and stored
at room temperature. Four hundred mesh copper–rhodium grids
(maxtaform) with a homemade carbon layer were glow discharged in air
for 1.5 min at medium power in a Harrick PDC-002 plasma cleaner. The
20 g L–1 sample was diluted (1/125 or 1/625), and
8 μL was incubated on the grids for 1 min before blotting (Whatman
filter paper No. 50). The grids were washed with water (three times)
and 2% w/v uranyl acetate (three times). After the last dose of uranyl
acetate was applied, the grid was left to incubate for 5 min before
blotting. A single-tilt room temperature holder in an FEI Tecnai G2
Spirit TWIN transmission electron microscope equipped with a tungsten
emitter at 120 kV was used. Images were recorded with an Eagle CCD
camera under low-dose conditions. The micrographs were binned two
times resulting in a pixel size of 2.2 Å per pixel at specimen
level.
7
Negative-Stain TEM Imaging of ELdcR
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Negative-stain transmission electron microscopy (TEM) samples were prepared on 300mesh carbon coated copper grids (C300Cu) with negative staining. To that end, grids were rendered hydrophilic by plasma treatment using a PDC-002 plasma cleaner (Harrick Plasma, USA). Low power setting (7.2 W applied to the RF coil) for 20 s.
Then, 15 μL of the pre-incubated ELdcR solutions (1 h at 37 °C at a concentration of 25 µм), ultrapure water and uranyl acetate (1% w/v) were dropped on Parafilm ® strip over a pre-heated (37 °C) glass surface. Plasma treated grids were sequentially placed on the ELdcR drop for 90 s, on ultrapure water for 60 s, and finally, on the negative staining solution for another 60 s. Blotting filter paper was used to remove excess solution after every step by touching the edge of the grid.
Images were obtained using a Tecnai Thermoionic T20 microscope operating at 200 kV (SAI, University of Zaragoza, Spain).
Then, 15 μL of the pre-incubated ELdcR solutions (1 h at 37 °C at a concentration of 25 µм), ultrapure water and uranyl acetate (1% w/v) were dropped on Parafilm ® strip over a pre-heated (37 °C) glass surface. Plasma treated grids were sequentially placed on the ELdcR drop for 90 s, on ultrapure water for 60 s, and finally, on the negative staining solution for another 60 s. Blotting filter paper was used to remove excess solution after every step by touching the edge of the grid.
Images were obtained using a Tecnai Thermoionic T20 microscope operating at 200 kV (SAI, University of Zaragoza, Spain).
8
Covalent Immobilization of Antimicrobial Peptides on Gold Surfaces
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To study the AM-ELR as a covalent coating, GL13K, VC, and GVC were covalently immobilized onto model gold surfaces (Figure 1) via the Cys residues present in the AMP/ELR/AM-ELR. 56 (link) To prepare the gold surfaces, cover glasses with a diameter of 12 mm (ThermoScientific) were cleaned with Argon plasma for 15 min at a high power setting (29.6 W applied to the RF coil) using a PDC-002 plasma cleaner (Harrick Plasma, USA). These cover glasses were then covered with a 40 nm gold layer using a sputter coater (Emitech K575X) with a gold layer with a purity of 99.99% (150 s, 30 mA). The resulting surfaces were immediately immersed in 200 μM AMP/ELR/AM-ELR solutions for 4 h, then washed three times with ultrapure water and ethanol to remove physisorbed molecules, dried under vacuum overnight and stored at -80 °C for further use.
9
Characterizing Plastic Particle Morphology via FE-SEM
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The surface morphology of the plastic samples and the shape of the plastic particles were examined using FE-SEM. Untreated and treated plastic samples were washed with fresh DI water two times and dried in a desiccator box. To observe the plastic particles, glass slides (Sail Brand) were washed with acetone (Sigma) and DI water, treated with oxygen plasma (Plasma Cleaner PDC-002, Harrick Plasma) for 1 min, and then immersed in DI water for 2 min to increase its hydrophobicity. Thereafter, for each sample, 10 μL of a solution containing the sample particles was dropped on the glass slides and dried. The dried samples and glass slides were immobilized on a sample holder with carbon tape and sputter-coated with a 5 nm thick platinum film using a JFC-1600 Auto Fine Coater (JEOL; operating settings: 30-mm distance, 20 mA, 40 s). FE-SEM imaging (JSM-7600F Schottky FE-SEM, JEOL) was performed at an accelerating voltage of 5.00 kV under various magnification levels between × 5000 and × 200,000 to capture particle in the nanometer scale.
10
Fabrication of Insulin-Loaded Silk Microneedles
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For the fabrication procedures, first we prepared the silk fibroin solution with insulin by mixing insulin solution (weight ratio as 33.98%), DI water (weight ratio as 50.98%), purple food dye solution (weight ratio as 8.50%) and silk fibroin (weight ratio as 6.54%). We assume that the densities of insulin solution, DI water, and dye solution are 1.0 g/cm3. Then we did the hydrophilic treatment of the microneedle mold using oxygen plasma (at 1000 mTorr for 1 min, Plasma Cleaner, PDC-002, Harrick Plasma, Ithaca, NY, USA). We put the microneedle mold in a vacuum chamber to facilitate the solution deposition in the following step. After that, we dropped 300 μL of silk fibroin solution with insulin on the microneedle mold. We put the mold into a chamber with silica gel as a desiccant for deposition. After the solution was fully dried, we put the microneedle mold in a vacuum chamber again to facilitate the deposition of OSTE later. We then dropped 1.0 g OSTE on the microneedle mold and made it cure at room temperature for 24 h. When it was fully cured, we removed OSTE with dried silk fibroin from the microneedle mold. Figure 1 shows the detailed fabrication procedures.
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