Bicinchoninic acid protein assay kit

We used 5‐ALA hydrochloride (COSMO BIO), which was dissolved into DMEM at various concentrations. In some experiments, we used the ABCG2 transporter inhibitor Ko143 (Sigma‐Aldrich), which were stored at −20°C after dissolution in DMSO. Polyclonal antibodies against ABCG2 (Cell Signaling Technology) and peptide transporter 1 (PEPT1) were purchased from Bioss Inc. The bicinchoninic acid protein assay kit was obtained from Wako Pure Chemicals.

Melphalan, adriamycin, vincristine, RPMI1640 medium, pepstatin, leupeptin, calpain inhibitor, phosphatase inhibitor cocktail I/II, and phenylmethylsulfonyl fluoride were purchased from Sigma (St. Louis, MO, USA). Dexamethasone, verapamil, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), Tris-HCl (pH 7.4), EDTA, NP-40, sodium orthovanadate, and bicinchoninic acid protein-assay kit were purchased from Wako (Tokyo, Japan). Dasatinib was purchased from ChemieTek (Indianapolis, USA). Fetal bovine serum, penicillin, and streptomycin were purchased from Gibco (Carlsbad, CA, USA).
Melphalan, Dexamethasone, and Dasatinib were soluble in dimethyl sulfoxide, attenuated in phosphate-buffed saline (PBS); pH 7.4, and filtrated through 0.45 μm syringe (Iwaki Glass, Tokyo, Japan) filter before use. adriamycin and vincristine were soluble in PBS. verapamil was soluble in ultrapure water, and filtrated through 0.45 μm syringe (Iwaki Glass) filter before use.

Using a Bicinchoninic Acid Protein Assay Kit (FUJIFILM Wako Pure Chemicals), the extract and reagents were reacted at 37 °C for 30 min, and the absorbance at 562 nm was measured using an iMark microplate reader (Bio-Rad, Tokyo, Japan). We determined the total protein content in EVs. The standard curve was prepared using serial dilutions of 2 mg/mL albumin solution from bovine serum. The estimated amount of EVs was evaluated based on the acetyl-CoA acetylcholinesterase activity, which was determined using a FluoroCet Exosome Quantitation Kit (System Biosciences, Palo Alto, CA, USA) according to the manufacturer’s instructions. In brief, after adjusting the protein concentration to 10 ng/μL, it was allowed to react with lysis buffer on ice for 30 min and mix with 0.5 M acetylcholine chloride at room temperature for 20 min in the dark. Fluorescence (measurement wavelength: excitation 530 nm/emission 585 nm) was measured using a Mithras LB940 plate reader (Berthold, Tokyo, Japan). The standard curve was created using serially diluted FluoroCet standard, which was supplied within the kit.