Polypropylene centrifuge tube

For the main e-liquid components, United States Pharmacopeia Grade (USP-grade) PG, USP-grade VG and 99% pure nicotine measured via gas chromatograph (GC-grade nicotine) (Sigma-Aldrich, St. Louis, MO) were used. PG and VG were mixed in volumetric proportions using 5 mL or 10 mL pipettors (Gilson, Middleton, WI) in clean 15 mL polypropylene centrifuge tubes (Corning Inc., Corning, NY). No further additives, such as flavors, were used. Amounts were verified by gravimetry using an analytical balance (Model AB54-S Mettler Toledo, Columbus, OH) with an accuracy of ± 0.4 mg. Nicotine concentration was 0.30 mg/mL.

Isotonic and pH-neutral solutions containing 20, 50, and 100 mg/mL of HCQS were prepared in volumetric flasks. Then, they were transferred to 50 mL of polypropylene centrifuge tubes (Corning, Corning, NY, USA) and stored in darkness at ambient temperature until use. The osmolality of the solutions was measured with a K-7000 vapor pressure osmometer (Knauer, Berlin, Germany). The cell and head temperatures were set as 60 °C and 62 °C, respectively, and allowed to stabilise for an hour before use. These temperatures followed those recommended in the instrument manual for calibrating and measuring sodium chloride aqueous solutions [49 ]. The measurement time and gain were 1.5 min and 16, respectively. Approximately 1 mL of each sample solution was drawn into glass microsyringes and inserted into the osmometer. One droplet from each sample was dispensed onto the thermistor for each osmolality measurement. The droplet was replaced by a new one when repeating the measurement. The experiments were conducted in quadruplicate (n = 4) for each HCQS solution. The target osmolality range was 260–360 mOsmol/kg H₂O [50 (link),51 (link)]. The pH of the solutions was measured with a pH 700 benchtop meter (Oakton, Vernon Hills, IL, USA). The target pH range was 6.8–7.5 [50 (link),51 (link)].

For RNA extraction, immature seeds were harvested over the course of several weeks. The individual seeds were pooled and sorted by weight. Following this, each seed was dissected, separating the cotyledon and embryo from the seed coat, and placed in separate 15-ml polypropylene centrifuge tubes (Corning, Acton, MA). All tissue was then frozen in liquid nitrogen and placed in storage at −80°C until it could be lyophilized. RNA of seed coats of seeds from the 50–100 mg, 100–200 mg and 400–500 mg seed weight stages of each isoline was extracted separately using the RNA for 5 ml volumes from ∼30 mg (50–100 mg and 100–200 mg seed weight stage) and ∼70 mg (400–500 mg seed weight stage) dry weight. The modified protocol used here is based on the protocols of McCarty [64] . Extraction of RNA from seed coats with dark pigmentation requires the use of a modified extraction protocol which prevents procyanidins from binding the RNA [65] (link), [66] (link). RNA was extracted from 200 mg of freeze-dried seed coats by using phenol chloroform extraction and lithium chloride precipitation supplemented with PVPP (polyvinylpyrrolidone), polyproline and BSA (bovine serum albumin). Library construction and high-throughput sequencing was carried out using RNA-Seq technology using Illumina GaII and HiSeq2000 instruments by Keck Center, University of Illinois.

Seed-free pulp (30 g) from each fruit was placed in 50-mL polypropylene centrifuge tubes (Corning^®, Tewksbury, MA, USA). The tube was subjected to centrifugation (4000× g, 4 °C, 10 min, Model SL 40R, Thermo Fisher Scientific, Langenselbold, Germany). The supernatant was filtered in the dark through 150 mm Whatman paper grade 4 (item 1009150, GE Healthcare Life Sciences, Little Chalfont, UK). The pellets were removed, and the clarified supernatant from the pulp of each fruit was used as a clarified juice extract.

The cells were harvested and resuspended at 1 × 10⁶/ml in prewarmed DMEM (Life technologies, USA) containing 2% fetal calf serum (Kang Yuan Biology, China) and 10 mM HEPES (Dalian Meilun Biology Technology co., LTD, China). The cell suspension was gently mixed in 15 ml polypropylene centrifuge tube (Corning, USA). And then Hoechst 33342 (Invirtrogen, USA) was added to a final concentration of 5 ug/ml and verapamil (Sigma-Aldrich, USA) of 80 ug/ml to control group. The cells were incubated for 90 minutes at 37°C and mixed every 30 minutes. After incubation, the cells were centrifuged at 500 g for 5 minutes, and resuspended in cold HBSS. Propidium iodide (Sigma-Aldrich,USA) was added into each tube at concentration of 1 ug/ml to discriminate dead cells. In order to avoid the effluxion of Hoechst 33342, further tests were performed at 4°C. SP cells were tested using flow cytometry and analyzed using Summit 5.2 software (Beckman Coulter, Inc., USA). The assays were conducted in triplicate and repeated at least 3 times.