All the reagents were used as received. The photoresists: SU-8 5 and S1808 as well as the photoresist developers were purchased from MicroChem. Cytop and the Cytop solvent were obtained from AGC. N-methylpyrrolidinone and ethylene glycol (EG) were purchased from Fischer Scientific. 1-Ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC), N-hydroxysulfosuccinimide (sulfo-NHS) and sulfo-NHS-biotin were purchased from Thermo Scientific. Acetone was obtained from VWR, ethanol from Gold Shield, hydrochloric acid from J. T. Baker and nitric acid from Macron Chemicals. PBS 10 × was obtained from Affymetrix and diluted by 10 with mQ water prior to utilization. Hellmanex, tetraethyl orthosilicate (TEOS), sodium nitrate, (3-aminopropyl)trimethoxysilane (APTMS) and poly(L-glutamic acid) (PGlu) (MW = 50 000-100 000) were purchased from Sigma Aldrich. The aqueous solutions were prepared using milliQ water. The gold nanoparticles (AuNPs) were synthesized using the well-known Turkevich method. The amino-terminated ssDNA sequences were obtained from IDT. Alexa647-streptavidin was purchased from Molecular Probes and Human α-thrombin was obtained from Haematologic Technologies. The glass slides used for the deposition of Cytop were obtained from Fischer. The nickel pellets used for the thermal evaporation were purchased from Kurt J. Lesker.
Hellmanex
Manufactured by Merck Group
Hellmanex is a laboratory cleaning agent designed to effectively remove organic and inorganic contaminants from various laboratory equipment and glassware. It is a concentrated, non-abrasive solution that can be diluted with water for use.
Automatically generated - may contain errors
Lab products found in correlation
Peg5000 pe, by Avanti Polar Lipids (2 mentions)
Biotin dppe, by Avanti Polar Lipids (2 mentions)
Dgs nta ni, by Avanti Polar Lipids (2 mentions)
Acetone, by Avantor (1 mentions)
Ethylene glycol, by Thermo Fisher Scientific (1 mentions)
Sulfo nhs biotin, by Thermo Fisher Scientific (1 mentions)
3 aminopropyl trimethoxysilane aptms, by Merck Group (1 mentions)
N methylpyrrolidinone, by Thermo Fisher Scientific (1 mentions)
Human α thrombin, by Prolytix (1 mentions)
Su 8 5, by MicroChem (1 mentions)
Nitric acid, by Merck Group (1 mentions)
Silver nitrate, by Merck Group (1 mentions)
D glucosamine hydrochloride, by Merck Group (1 mentions)
Rpmi 1640 media, by Merck Group (1 mentions)
Fetal calf serum (fcs), by Thermo Fisher Scientific (1 mentions)
Poly l lysine (pll), by Merck Group (1 mentions)
Neon transfection system, by Thermo Fisher Scientific (1 mentions)
5 protocols using hellmanex
1
Immobilization and Detection of ssDNA on Cytop Surfaces
2
Fabrication of Nanomaterial Devices
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PFM was purchased from Apollo Scientific Ltd. (Stockport, U.K.). D-(+)-Glucosamine hydro-chloride, sodium dodecyl sulfate (SDS), Hellmanex, nitric acid, and silver nitrate were purchased from Sigma-Aldrich. Ethanol was purchased from Scharlab (Barcelona, Spain), a Sylgard 184 kit was purchased from Ellsworth Adhesives (Madrid, Spain) and Si-wafers were purchased from Wafer World (West Palm Beach, FL, USA).
3
Supported Lipid Bilayer Formation for Immune Cell Studies
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SLBs were formed on glass-bottomed 96-well plate (DOT Scientific Inc, #MGB096-1-2-LG-L). Briefly, the plate was cleaned with 2.5% Hellmanex (Sigma-Aldrich, #Z805939-1EA), etched with 5 N NaOH, and used for SLB formation as previously described (Ahrends et al., 2017 (link)). Briefly, small unilamellar vesicles (SUVs) derived from dried lipid film containing 95.5% POPC (Avanti Polar Lipids, #850457C), 2% biotin-DPPE (Avanti Polar Lipids, #870285P), 2% DGS-NTA-Ni (Avanti Polar Lipids, #790404C), and 0.1% PEG 5000-PE (Avanti Polar Lipids, #880230C) were added onto freshly treated plates to form SLBs. The SLBs were rinsed with wash buffer (1× PBS containing 0.1% BSA) and mixed with 1 μg/ml streptavidin, 0.1 nM His-tagged human PD-L1 ECD, and 3 nM His-tagged human ICAM-1 ECD at 37°C for 1 hr. Afterward, the SLBs were rinsed with wash buffer and further incubated with 5 μg/ml biotin anti-human-CD3ε at 37°C for 30 min, followed by three rinses with wash buffer and three rinses with imaging buffer (20 mM HEPES pH 7.5, 137 mM NaCl, 5 mM KCl, 1 mM CaCl2, 2 mM MgCl2, 0.7 mM Na2HPO4, 6 mM D-glucose).
4
Transient Transfection of Jurkat T Cells
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Jurkat T cells in RPMI-1640 media (Sigma-Aldrich), complemented with glutamine and 10% fetal calf serum (Life Technologies), were grown in an incubator under controlled conditions of 37°C, 5% CO2, and 100% humidity. The cells were transiently transfected using the Neon® transfection system (Life Technologies). One microgram of vector DNA per shot (3 pulses of 1325 V lasting for 10 ms) per 200,000 cells was used (see manufacturer’s instructions). Twenty-five-millimeter diameter microscope coverslips were cleaned by incubation with 2% Hellmanex (Sigma-Aldrich) overnight at 42˚C and subsequently washed with MiliQ water. Prior to use, the coverslips were coated with poly-l -lysine (Sigma-Aldrich). Twenty hours after transfection, the cells were washed with PBS, resuspended in phenol red-free RPMI-1640 media (Sigma-Aldrich), seeded on the poly-l -lysine-coated coverslips, and incubated for 5 min at 37°C under 5% CO2. After a quick PBS wash the cells were fixed using 4% paraformaldehyde in PBS at 37°C for 10 min. After removal of excess liquid, the fixation was stopped with 0.1 M NH4Cl in PBS and the cells were washed with PBS. Finally, the coverslip was placed into a ChamLide holder for imaging (Live Cell Instruments).
5
Supported Lipid Bilayer Functionalization
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SLBs were formed on glass-bottomed 96-well plate (DOT Scientific Inc, #MGB096-1-2-LG-L). Briefly, the plate was cleaned with 2.5 % Hellmanex (Sigma-Aldrich, #Z805939-1EA), etched with 5N NaOH, and used for SLB formation as previously described 57 . Briefly, small unilamellar vesicles (SUVs) derived from dried lipid film containing 95.5% POPC (Avanti Polar Lipids, #850457C), 2% biotin-DPPE (Avanti Polar Lipids, #870285P), 2% DGS-NTA-Ni (Avanti Polar Lipids, #790404C) and 0.1% PEG 5000-PE (Avanti Polar Lipids, #880230C) were added onto freshly treated plates to form SLBs. The SLBs were rinsed with wash buffer (1x PBS containing 0.1% BSA) and mixed with 1 μg/mL streptavidin, 0.1 nM His-tagged human PD-L1 ectodomain, and 3 nM His-tagged human ICAM-1 ectodomain at 37 °C for 1 h. Afterward, the SLBs were rinsed with wash buffer and further incubated with 5 μg/mL biotin anti-human-CD3ε at 37 °C for 30 min, followed by three rinses with wash buffer and three rinses with imaging buffer (20 mM HEPES pH 7.5, 137 mM NaCl, 5 mM KCl, 1 mM CaCl 2 , 2 mM MgCl 2 , 0.7 mM Na 2 HPO 4 , 6 mM D-Glucose).
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