Nanoorange protein quantitation kit

4 mL of stock culture samples (∼1 × 10⁹ CFU/ml) were used to determine the total protein content using the NanoOrange^® Protein Quantitation Kit (Molecular Probes, Inc., Eugene, OR, United States) following the manufacturer’s instructions. The average cell protein content was determined as a ratio of total protein content (ppm protein) to the cell concentration (CFU/mL). Cellular protein content of calorimetric batch cultures in lag and exponential growth phase were below detection limit (10 ng/ml) and therefore not reported here.

Morphology of the isolated extracellular vesicles was assessed by transmission electron microscopy (TEM) as described previously [17 (link)]. The initial volumes of plasma or blood for the study of EV using the TEM were 20 mL.
The size and concentration of EVs were determined by NTA using the NanoSight NS300 (Malvern, UK) as described previously [16 (link)].
To evaluate the protein concentration of exosomes, a NanoOrange Protein Quantitation kit (NanoOrange^® Protein Quantitation Kit, Molecular Probes, Eugene, OR, USA) was used in accordance with the manufacturer’s recommendations.
Quantitative analysis of the exosomal tetraspanines on the surface of the isolated extracellular vesicles was carried out using flow cytometry as described previously [46 (link)]. Flow cytometry was performed on the Cytoflex (Becman Coulter, BioBay, China), using CytExpert 2.0 Software. The MFI of stained exosomes was analyzed and compared to the isotype control (BD bioscience, Heidelberg, Germany).

Ten ml of E. coli suspension (approximately 10⁸/ml) grown in the presence of 1 mM IPTG was centrifuged at 4400 × g for 10 min at 4°C, and the supernatant was removed. The pelleted cells were resuspended in 1 ml of sterile column buffer (50 mM Tris, pH 8, 300 mM NaCl, 50 mM imidazole) and were ruptured by sonication in 3 pulses of 30 s each. Then, the bacterial fragments were removed by centrifugation (7000 × g, 15 min). The supernatant was collected, diluted with column buffer and applied onto the affinity column (His-Bind Ni Column, Novagen) previously equilibrated with the column buffer. The column was washed with the same buffer, and then LLO was desorbed with the elution buffer (50 mM MES, pH 6, 300 mM NaCl, 250 mM imidazole). The collected fraction was subsequently dialysed to remove imidazole (50 mM MES, pH 6, 300 mM NaCl, 5 mM cysteine HCl). Protease inhibitors: fluoro 4-(2-aminoethylo)-benzenesulphonyl.HCl (AEBSF, Sigma), EDTA (Sigma), and glycerol (Merck) were added to a final concentration of 1 mM, 10 mM, and 15% (v/v), respectively. Protein concentrations were assayed with NanoOrange® Protein Quantitation Kit (Molecular Probes) using an infinite M200 PRO reader (TECAN, Goring on Thames, UK).

Each CPS was subjected to rigorous quality control tests as previously described (25 (link)) on 1-mg/ml samples. Nucleic acids were quantified using an ND 1000 spectrophotometer (NanoDrop, Wilmington, DE). The absorbance was measured at 230 and 260 nm on CPS samples. Calculations were done with the NanoDrop software. According to the manufacturer, results are reproducible between 2 and 100 ng/μl. Proteins were quantified on CPS samples using the modified Lowry protein assay kit (Thermo Fisher Scientific, Saint-Laurent, Quebec, Canada) in a 96-well microplate according to the manufacturer’s instructions. The calculated limit of detection (P ≤ 0.05) was 5 μg/ml for the modified Lowry assay. Proteins were also quantified on CPS samples using a NanoOrange protein quantitation kit (Molecular Probes, Eugene, OR) according to manufacturer’s instructions by heating samples into microtubes, followed by transfer to a 96-well microplate for fluorescence reading. The calculated limit of detection (P ≤ 0.05) was 4 μg/ml for the NanoOrange assay.

Exosomes from blood plasma (from 18 mL of venous blood) and ascites (about 18–20 mL) were isolated using ultrafiltration with ultracentrifugation as described previously [39 ]. Exosome samples (in 400 µL of PBS) from plasma and ascites fluid were aliquoted and stored either at −80 °C or in liquid nitrogen. The aliquots were thawed once before use.
Morphology of the isolated EVs from plasma and ascites was assessed by TEM as described previously [6 (link)]. The volumes of exosomes from plasma or ascites for the study of EV using the TEM were 15 µL.
To evaluate the exosomal protein concentration, a NanoOrange Protein Quantitation kit (NanoOrange^® Protein Quantitation Kit, Molecular Probes, Eugene, OR, USA) was used in accordance with the manufacturer’s recommendations. The volumes of exosomes from plasma or ascites for protein quantification were 8 µL.
Analysis of CD9/CD63/CD81/CD24 subpopulations in plasma and ascites fluid exosomes was carried out using flow cytometry as described previously [8 (link)]. The volumes of exosomes from plasma or ascites for flow cytometry were about 130 µL (30 μg exosomal protein). The MFI of stained exosomes was analyzed and compared to the isotype (BD bioscience, Heidelberg, Germany) and negative controls.