Chloroform

Static (SLS) and dynamic (DLS) light scattering were used to study the solution behavior of copolymers with different compositions by Photocor Complex instrument (Photocor Instruments Inc., Moscow, Russia). The light source was the Photocor-DL diode laser with the wavelength λ = 659.1 nm and controllable power up to 30 mW. Procedure was described early [33 (link)].
As is known, the behavior of amphiphilic polymers in solutions strongly depends on the thermodynamic quality of the solvent with respect to the components. Therefore, the choice of solvents for research is an important task. As can be seen from Table 1, the blocks of the considered brushes (MI, PtBMA, PMAA) dissolve in different ways in the solvents we have chosen.
The synthesized precursors and molecular brushes (PB, APB), were molecularly dissolved in chloroform. Therefore, their molar mass characteristics were determined in chloroform (Sigma-Aldrich, Saint Louis, MO, USA)
The molar mass of the PF macroinitiator were measured in chloroform, which is a thermodynamically good solvent for PF.

Silicon wafers (p-type, {100}, 1 mm thickness) were obtained from MicroChemicals GmbH and cleaned with acidic piranha solution (30% H₂O₂ and concentrated H₂SO₄ in a 1 : 3 ratio) before use (Caution: acidic piranha solution may react with organic compounds to form an explosive mixture). Gold (111)-coated slides were acquired from Arrandee and cleaned with ethanol before use. 1,2,dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and cholesterol were obtained from Avanti Polar Lipids, Inc. Lipid solutions were prepared in chloroform or chloroform : methanol (9 : 1); both solvents were HPLC grade and obtained from Sigma-Aldrich. Calcium chloride was obtained from Sigma Aldrich (purity > 99.9%) and stored under argon and potassium chloride was obtained from Alfa Aesar (purity 99%). Water purified with a tandem Milli-Q Gradient A10 system (Millipore, France, resistivity 18 MΩ cm, TOC < 5 ppb) was used throughout.

Cer and C1P measurements were achieved as described in Bini et al. [39 (link)]. Briefly, cells were labeled with 40 μCi/ml of [³H] palmitate (Perkin Elmer, Connecticut, USA) for 24h in serum–free medium in the presence or absence of agonists. Cells were then washed with ice-cold calcium-free phosphate-buffered saline (PBS) and scraped into methanol and chloroform (2:1) (Sigma Aldrich, Milan, Italy). Lipids were extracted by separation of phases as described in [39 (link)]. chloroform phases were dried, and lipids were separated by thin-layer chromatography (TLC) using silica gel 60-coated glass plates (Merck, Darmstadt, Germany). The plates were developed for 50% of their lengths with chloroform/methanol/acetic acid (9:1:1, v/v/v) and then dried. They were then developed for their full length with petroleum ether/diethylether/acetic acid (60:40:1, v/v/v) (Sigma Aldrich, Milan, Italy). The position of Cers was identified after staining with I₂ vapor by comparison with authentic standards. Radioactivity of the samples, obtained by scraping the Cer and C1P spots from the plates, was quantified by liquid scintillation counting. CerK activity was assayed as described previously [39 (link), 47 (link)] with some modifications.

⁸⁹Zr-oxalate stock (2–40 MBq; Perkin Elmer) was diluted to 500 μL with HPLC-grade water and neutralised with 1 M NaOH (Sigma-Aldrich). To this was added 20 μL of a 10-mg/mL solution of 8-hydroxyquinoline (ACS reagent, 99%, Sigma-Aldrich: 252565) in chloroform (Sigma-Aldrich), in a screw-top round-bottomed tube (BRAND® culture tubes, 6.5 mL, AR-Glas®, Sigma-Aldrich) and held onto a vortex mixer (Grant Bio, model PV-1) for 5 min using a clamp. chloroform was added (480 μL) before vortexing for 25 min. After brief centrifugation, the chloroform phase containing Zr-oxine was removed and evaporated at 80 °C in a conical bottom HPLC vial (Supelco CD vial, 9-mm screw, Sigma-Aldrich), before resuspension in 15 μL of dimethyl sulfoxide (DMSO anhydrous, Sigma-Aldrich) at 50 °C for 20 min. Radiochemical yield (RCY) was calculated as the percentage of total original activity extracted into the chloroform phase, giving an average RCY of 74.2% ± 4.3 SEM (n = 16). Negative control synthesis was performed without addition of 8-hydroxyquinoline to the chloroform phase, resulting in retention of all the activity in the aqueous phase.

Ethanol and chloroform were obtained from Sigma-Aldrich (Germany). Pyridine was purchased from Chempur (Poland), and ethylchloroformate was obtained from Fluka (Switzerland). As the internal standard (IS), the compound 4-chloro-L-phenylamine (Sigma-Aldrich, Germany) in chloroform was used. All chemicals were pure and of HPLC solvent grade.

LucEV71 replicon or pCMV6-PHB (Origene)-harbouring E. coli was grown in LB broth supplemented with kanamycin (50 μg/mL). Both plasmids were extracted using QIAprep Spin Miniprep Kit (Qiagen). LucEV71 was linearized with Mlu1 restriction enzyme (R0198S, New England Biolabs). The linearized plasmid was then purified using chloroform/phenol/isoamyl (CPI 24:25:1) and chloroform (Sigma Aldrich). Purified linear plasmid (1 μg) was then subjected to in vitro transcription using MEGAscript T7 Transcription Kit (AM1334, ThermoScientific), according to the manufacturer’s instructions. The RNA product was further cleaned-up with chloroform/phenol/isoamyl (CPI 24:25:1) and chloroform. NSC-34 and SK-N-SH cells (2.5×10⁴ cells) seeded in a 96-well plate were reverse-transfected with 50nM siPHB, siNTC or left untreated for 48 hours. At 48 h.p.t. 1 μg of lucEV71 RNA was added into each well and incubated for 48 hours. The luminescence signal was then captured using Nano-Glo Luciferase Assay System (Promega, N1103). Signal was normalized against non-transfected cells. The pCMV6-PHB plasmid (0.5 μg) was transfected into NSC-34 cells for 48 hours prior to viral infection. The culture supernatant was harvested at 48 h.p.i. for determination of viral titer by plaque assay.