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Tim4-coated microplates (Cat#: 79201, Wako Pure Chemical Industries, Ltd.) were incubated with 100 μL/well of cell culture supernatants for 2 h at room temperature with agitation. After washing, 100 μL of HRP-labeled anti-CD63, -rBC2LCN (0.1 μg/mL), -R-10G (0.1 μg/mL) were reacted for 1 h at room temperature with agitation. Finally, TMB solution was added and developed for 30 min at room temperature. The reaction was stopped by adding 100 μL of 1 N HCl and detected dominant absorbance at OD450 and secondary wavelength at OD620.
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