Endospecy es 50m kit

A United States Pharmacopeia Reference Standard Endotoxin (U.S. Pharmacopeial Convention, Rockville, MD, USA) was used in this study. Endotoxin-free plastic instruments and the Endospecy® ES-50M kit were purchased from the Seikagaku Corporation (Tokyo, Japan); the chromogenic synthetic substrates Boc-Leu-Gly-Arg(LGR)-pNA, Boc-Val-Pro-Arg(VPR)-pNA and Boc-Met-Thr-Arg(MTR)-pNA (Boc, tert-butoxycarbonyl; para-nitroanilide, pNA) were purchased from the Peptide Institute (Osaka, Japan); protease inhibitors leupeptin and pepstatin A were purchased from Merck KGaA (Darmstadt, Germany); water for injection was purchased from Otsuka Pharmaceutical (Tokyo, Japan); the eight injectable drugs were purchased from Otsuka Pharmaceutical or FUSO Pharmaceutical Industries (Osaka, Japan); the protein assay kit was purchased from Bio-Rad (Hercules, CA, USA); and all other reagents for the biochemical assays were purchased from Sigma-Aldrich Japan (Tokyo, Japan).

Protein concentration in the MV suspensions was determined using a Bradford assay, with bovine serum albumin as the standard. Lipid concentration in the MV suspensions was determined using FM4‐64 dye (Life Technologies, Carlsbad, CA, USA), with water‐soluble linoleic acid (Sigma, St. Louis, MO, USA) as the standard. Standards and samples (5 µl) were mixed with 100 µl of 5 µg ml⁻¹ FM4‐64 in the 96‐well black plate, and incubated for 10 min at room temperature. Fluorescence from FM4‐64 (excitation at 535 nm and emission at 625 nm) was detected with the plate reader Cytation5 (Biotek, Winooski, VT, USA). Limulus assay was performed to quantify LPS using an Endospecy ES‐50M kit (Seikagaku Co., Tokyo, Japan) according to the manufacturers’ instructions, with LPS from Escherichia coli O111:B4 (Sigma) as the standard. MV particle size was measured from TEM images using the Fiji image processing package (a variant of ImageJ) (Schindelin et al., 2012).