Quantipro bca assay kit

Size, morphology and purity of Exos were observed before and after HPH treatment to validate the stability of vesicles.
For Cryo-TEM analysis, 3 μL of sample were directly dropped onto Formvar/Carbon 200 mesh Cu Agar^® grids. Samples were observed in a Tecnai FEI^® TEM operated at 80 kV accelerating voltage.
The concentration and mean size of Exos were determined by recording and analyzing the Brownian motion of particles using a NanoSight NS300 system and NPs Tracking Analysis (NTA) 3.3-Sample Assistant Dev Build 3.3.203—Analytical Software (Malvern, Worcestershire, UK) according to the manufacturer’s protocol. Purified Exos were diluted 1:100 in 1 mL PBS at RT and monitored for 260 s with manual shutter and gain adjustments. The recorded videos were analyzed using a NPs Tracking Analysis software. Mean size of Exos diluted in water (0.8% wt/vol) was analyzed at 25 °C, using a Zetasizer Nano ZS (Model ZEN3600, Malvern Instruments Ltd., UK) equipped with a solid-state laser (λ = 633 nm) at a scattering angle of 173°. The cuvettes used are the 12-mm square glass cuvettes with square aperture for 90° sizing (Malvern; # PCS1115). The recovery of Exos was indirectly estimated by measuring the surface protein quantitation using the BCA assay. Total protein amount was quantified with QuantiPro^TM BCA Assay Kit (Sigma Aldrich; St. Louis, MO, USA) in each vesicle preparation.

Gelatin type B (Sigma‐Aldrich, USA, gel strength: ≈225 g Bloom), trans‐cinnamic acid (Alfa Aesar, USA, purity: 99+%), EDC (ACROS Organics, USA, purity: 95+%), lithium phenyl‐2,4,6‐trimethylbenzoylphosphinate (LAP, Sigma‐Aldrich, Germany, purity: 95+%), dopamine hydrochloride (Alfa Aesar, USA, purity: 99%), ammonium hydroxide solution (Sigma‐Aldrich, Germany, 28% NH₃ in H₂O, 99.99+% trace metals basis), curcumin (Sigma‐Aldrich, Germany, purity: 98%), glycine (ACROS Organics, USA, purity: 99%), sodium bicarbonate (Sigma‐Aldrich, Germany), 2,4,6‐trinitrobenzene sulfonic acid (CSL, AU), QuantiProTM BCA assay kit (Sigma‐Aldrich, USA), bovine serum albumin (BSA, Sigma‐Aldrich, USA), 2,2‐diphenyl‐1‐picrylhydrazyl (Alfa Aesar, USA, purity: 95% ), Riboflavin (Sigma‐Aldrich, Germany, purity: 98+%), NBT (Sigma‐Aldrich, Germany, purity: 90+%), l‐methionine (Sigma‐Aldrich, Germany, purity: 98+%), Dulbecco's modified Eagle's medium/high glucose (DMEM/HG, HyClone, USA), DAPI staining solution (Abcam, USA), Alexa FluorTM Phalloidin (Invitrogen, USA), and 2′,7′‐dichlorofluorescin diacetate (Sigma‐Aldrich, USA).

Hundred mL aliquots from Control, +ASTM and +Infochemicals algal cultures were centrifuged at 4500 × g for 15 min at 4°C to extract sEPS. Supernatant was first passed through a 0.22 μm pore-size filter and dialyzed against distilled water using a SnakeSkin Dialysis Tubing (3.5 kDa MWCO, Thermo Scientific). After dialysis the sEPS were freeze-dried (CoolSafe, ScanVac, LaboGene) and re-suspended in 1200 μl of HPLC grade water for further quantification assays. Carbohydrates were measured using the anthrone method using glucose as a reference standard (Le and Stuckey, 2016 (link)). Proteins were measured using the BCA assay kit (QuantiPro^TM BCA Assay Kit, Sigma Aldrich) using bovine serum albumin (BSA) as a reference standard (Georgiou et al., 2008 (link)). Fatty acids were measured using the method reported by Kapoore (2014) . The concentrations of each fraction were normalized by the algal cells’ concentration.

Proteins were extracted from at least two biological repeats of each of the life-cycle stages, namely tachyzoites (~10⁸), cysts (~10⁷) and oocysts (~10⁷). Briefly, each biological sample was homogenized with 5 volumes glass sand and lysed in 200 μl radioimmunoprecipitation assay (RIPA) buffer (20 mM Tris-HCl pH 7.5, 150 mM sodium chloride, 2 mM EDTA, 1% DOC, 1% Triton X-100) containing phenylmethylsulfonyl fluoride (PMSF). Then, samples were sonicated (2%, 1 s ON and 1 s OFF cycle, 5 times, 8 repeats) and the supernatant containing total soluble proteins was collected after 20-min centrifugation at 13,400 g. The concentration of the protein was determined by the protein quantitative kit (QuantiProTM BCA Assay Kit, Sigma).

The activity of NFATc1 was assessed on 3 μg nuclear extracts using the TransAM™ NFATc1 Transcription Factor Assay Kit (Active Motif, Carlsbad, USA), according to manufacturer’s instructions. A Nuclear Extract Kit (Active Motif) was used for nuclear extract preparation, and the protein content was measured with a QuantiProTM BCA Assay Kit (Sigma-Aldrich). The results (OD 450/655 nm) were reported as a percentage increase over control cells.
Time-course experiment to evaluate the best time of NFATc1 activation following 1 ng/mL RANKL stimulation was performed, including 6, 12 and 24 h. According to the results obtained, a stimulation time of 12 h was selected.

The BALF supernatant was centrifuged at 3000 rpm for 10 min at 4 °C, and the total protein concentration was measured using the QuantiProTM BCA Assay Kit (Sigma-Aldrich Inc.). The total protein concentration and cytokine levels were determined using the QuantiProTM BCA Assay Kit. A hematology analyzer was used to count the number of resuspended cells following resuspension in 50 μL PBS buffer.

Approximately 3000 L1 animals from the N2 strain were treated with 10 mg/mL or 50 mg/mL GHE or control solution for 72 h, until 1-day adulthood. Worms were then harvested with M9 buffer and sonicated with a homogenization buffer (5 mM Tris-HCl pH 8.0; 1% glycerol; 1 mM EDTA) plus 10 μL protease inhibitors (Amresco). The lysates were centrifuged at 20000 ×g for 30 min at 4°C. Protein was quantified using the QuantiProTM BCA Assay Kit (Sigma-Aldrich, St. Louis, MO, USA).
Proteasome activity was measured as “chymotrypsin-like activity,” using the peptide substrate succinyl-leu-leu-val-tyr-4-methylcoumaryl-7-amide (SLLVY-MCA) (Sigma-Aldrich, St. Louis, MO, USA). All measurements were performed in the presence and absence of 20 μM MG-132, a proteasome inhibitor. Enzyme kinetics were monitored in a multilabel microplate reader (PerkinElmer VICTOR X3, MA, USA), every 30 minutes for 1 h at 37°C, with excitation at 380 nm and emission at 440 nm. Relative proteasome activity was calculated as the difference between the total activity and the remaining activity in the presence of MG-132.