Amicon ultra 15 centrifugal filter 100 kda mwco

Before isolation, CM3D was concentrated by UF on the Amicon Ultra-15 Centrifugal Filter 100 kDa MWCO (Millipore Sigma) at 4000× g until 2–3 mL was left in the upper compartment. The remaining liquid was collected, the filter was rinsed twice with 1 mL of PBS, and PBS was added for a final volume of 10 mL. Exosomes were isolated from CM3D using the Automatic Fraction Collector (AFC; Izon Science) according to the manufacturer’s recommendations. Briefly, the enriched exosome solution was fractionated using a second generation qEV10/35 nm column (Izon Science). The 15 mL fraction range previously identified as containing exosomes was collected and concentrated at 1000× g using the Amicon Ultra-4 Centrifugal Filter 100 kDa MWCO (Millipore Sigma). The remaining solution was collected and the filter was rinsed twice with 150 µL of PBS to remove residual particles from the Amicon membrane and then to obtain a final volume of 800 µL of exosome solution. These exosomes solubilized in PBS were used to supplement the eAC culture medium (“qEV” conditions).

CM3D was collected and prepared as previously detailed. After concentration using the Amicon Ultra-15 Centrifugal Filter 100 kDa MWCO (Millipore Sigma), exosome isolation was performed on the second generation of qEV original/35 nm columns using the AFC (Izon Science). PKH26 labeling was performed with the PKH26 Red Fluorescent Cell Linker Kit (Sigma Aldrich) according to the manufacturer’s instructions. Briefly, exosomes were concentrated at 4000× g using the Amicon Ultra-4 Centrifugal Filter 100 kDa MWCO (Millipore Sigma) and washed twice with diluent C. After PKH26 labeling, exosomes were concentrated in the Amicon Ultra-4 Centrifugal Filter 100 kDa MWCO, fractionated with AFC to eliminate PKH26 micelles and concentrated again. After micro-BCA quantification to determine total protein concentration, exosomes labeled with PKH26 (PKH26-exos) were stored at −80 °C until use.