2 d quant kit

After incubation for 24, 36, or 48 h, the medium dosed with phenanthrene, pyrene, or benzo[a]pyrene was carefully removed by centrifugation at 4,000×g for 5 min at room temperature. Cells were washed twice with 30 mL of PBS (pH 7.6) and pelleted by centrifugation under the same conditions. For protein extraction, the cell pellets were resuspended in 2 mL lysate (7 M urea, 2 M thiourea, 4% w/v 3-[3-cholamidopropyl dimethylammonio]-1-pro-panesulfonate [CHAPS], 60 mM dithiothreitol, 2% v/v Pharmalyte 3–10, and a protease inhibitor cocktail) and sonicated on ice for 10 min using a 500 ms/s pulse at 40 W. The sonicated cell suspensions were then centrifuged at 10,000×g for 30 min at 4°C. Protein concentration was estimated using the 2-D Quant Kit (Amersham, USA).

The samples were briefly thawed on ice and dissected to remove meninges and exclude the blood vessels and the white matter. Cleaned samples were then homogenized at 50 mg tissue/ml in an ice cold cell lysis buffer (20 mM Tris–HCl, 150 mM NaCl, pH 7.5) (Cell Signaling Technology, Danvers, MA, USA). Protease inhibitor cocktail (Complete (Roche)) was added immediately before use. Protein concentrations were measured using a 2-D Quant kit (Amersham Biosciences, Piscataway, NJ, USA).

Adult E. granulosus worms were collected from the intestine by dissection microscopy, washed extensively with sterile PBS (Gibco, California, USA) containing 100 U/ml penicillin G, 100 μg/ml streptomycin (Gibco, California, USA) and cultured at a density of approximately 500 worms per ml serum-free RPMI 1640 medium (Gibco, California, USA) supplemented with 2 % glucose (Sigma-Aldrich, Missouri, USA) and antibiotics for 24 h at 37 °C.
To generate ES, the supernatant was harvested, centrifuged to remove eggs and worm debris and concentrated using a micro-concentrator with a 5 kDa cut-off (Millipore, Nassachusetts, USA). To prepare total adult worm antigen, the remaining adult worms from the culture were washed with PBS and homogenized by sonication. The homogenate was centrifuged (10000 g, 5 min) and the soluble fraction collected as AWA. ES and AWA were stored at −80 °C until used. A 2D Quant Kit (Amersham Biosciences, Buckinghamshire, UK) was used to determine the protein concentration in these preparations. The test for endotoxins was performed in AWA and ES, respectively. DCs showed the negligible expression of co-stimulatory molecules after stimulation with the same level of LPS, which suggested that the presence of LPS in purified antigens was lower than its effective concentration.

Cells were harvested (9000 rpm, 10 min, 4°C) at four specific time points (2, 4, 8, and 12 d) and washed four times with a suspension in PBS (10 mM, pH 7.8) pre-chilled at 4°C. Cells were then quickly frozen with liquid nitrogen, transferred to a pre-chilled pestle, and ground into powder using a mortar and pestle. The cell powder (0.2 g) was placed in a sterilized Eppendorf tube at 4°C and suspended in lysis buffer (0.4 mL) containing 8 M urea, 2 M thiourea, 4% (w/v) CHAPS, 75 mM NaCl, 50 mM Tris-HCl (pH 8.0), 2 mM phenylmethylsulfonyl fluoride, and 4 μL of a protease inhibitor cocktail. The suspended matter was shaken and mixed on a vortex for 30 s at 20 min intervals for 2 h. The lysed cells were centrifuged at 4°C and 13 200 rpm for 45 min to obtain the cleared cell extract. The supernatant was then stored at -80°C until further use. Before MS analysis, the protein concentrations of the supernatants were determined using a 2-D Quant kit (Amersham Biosciences, Piscataway, NJ).

The total protein was extracted by a modified phenol extraction method as described by Desjardin et al. (2012) [67 (link)]. The protein concentration was determined using 2D-QUANT kit (Amersham Biosciences, Little Chalfont, UK), then it was digested by trypsin (Promega, Madison, WI, United States). Desalting and concentration of the peptides were implemented before the operation of HPLC by NanoLC-Ultra (Eksigent, Dublin, OH, USA) coupled with an MS analysis by Qexactive PLUS (Thermo Fisher, Waltham, MA, USA). LC-MS analyses were performed on 3 independent batches: dry seeds, 16h-imbibed seeds and 30 h-imbibed seeds.

Leaf total proteins were extracted according to the method of Lv et al.21 (link) with minor modifications. Approximately 500 mg of fresh leaves from each biological replicate was ground into a fine powder in liquid nitrogen. The ground powder was suspended in 4 mL SDS buffer (30% sucrose, 2% SDS, 100 mM Tris-HCl, pH 8.0, 50 mM EDTA-Na₂, 20 mM DTT) and 4 mL phenol (Tris-buffered, pH 8.0) in a 10 mL tube, followed by the addition of 1 mM phenylmethanesulfonyl fluoride (PMSF) and 20 μL protease inhibitor cocktail (Merck KGaA, Germany) per 1 g of fresh sample to inhibit the protease activities. Samples were mixed vigorously for 15 min at room temperature, centrifuged twice at 15700× g for 15 min each, and the supernatants were precipitated with 100 mM cold ethanolamine-methanol solution at −20 °C over night. After centrifuging at 15700× g for 15 min, the pellets were rinsed with cold acetone (−20 °C) and further centrifuged three times. After freeze-drying, the pellets were added to 300 μL of solubilization buffer (7 M urea, 2 M Thiourea, 1% DTT (w/v) and 4% CHAPS) at room temperature for 2 h. Insoluble materials were removed by centrifugation at 15700× g for 15 min, and protein samples were measured using a 2-D Quant Kit (Amersham Bioscience, USA). The final protein solution was stored at −80 °C for later use.