Bca1 1kt kit

BCA1-1KT kit (Sigma, USA) was used for protein quantification assay. Lysate (Umibio, no. UR33101) was added to purified extracted exosomes and kept on ice for 30 min. The volume ratio of reagent A/reagent B equalled to 50 was prepared. The total volume was calculated as 200 µl for each well multiplied by the number of total samples including standard and test samples. The standard dilution method was adopted by adding deionized water (180 µl in tube 1). Twenty-five microliters of standard and test samples were added into 96-well plates with 200 µl BCA solution and mixed moderately. Plate was covered with aluminum foil and incubated at 37 for 30 min. The sample was analyzed by spectrophotometer and protein concentration was calculated from the standard curve (Pandey and Mann 2000) .

The protein content was determined using the BCA (bicinchoninic acid) protein assay using the BCA1-1KT kit (Sigma). The nucleic acid content was determined using the Quant-iT PicoGreen dsDNA kit (Invitrogen Molecular Probes). e. SDS-PAGE: Precast polyacrylamide 4 to 20% gradient gels (Bio-Rad) were used for SDS-PAGE analysis. The E. coli strain O111:B4 LPS was used again as reference. Samples were mixed 4:1 (v/v) with a sample loading buffer (66 mM Tris-HCl pH 6.8, 20% v/v glycerol, 2% v/v SDS, 5 mM EDTA, 14.3 M mercaptoethanol, 0.025% w/v bromophenol blue) and boiled for 10 min. 25 µg of the sample was loaded on a gel. Gels were run in a buffer composed of 50 mM Tris, 383 mM glycine and 10% SDS, at 100 V. LPS were detected by silver nitrate staining (Kittelberger et al. 1993 , Zhu et al. 2012) .
After this first coloration, SDS-PAGE was further stained with Stains All to detect anionic polysaccharides (Rigouin et al. 2009) .