Amicon ultra 2 30 kda mwco centrifugal filters

100 µg of freshly isolated HCT EVs or liposomes were incubated with 4 µL 1 mM Cy5 NHS‐Ester (Lumiprobe, Hunt Valley) at room temperature for 1 h. Unreacted dye molecules were removed by washing the samples with PBS using Amicon Ultra‐2 30 kDa MWCO centrifugal filters (Merck Millipore, Darmstadt, Germany). For labelling of EVs, an excess of fluorophores was used.
The labelling reaction was done in 100 µL at a working concentration of 40 µM Cy5‐NHS. That corresponds to 2.4 × 10¹⁵ available fluorophore molecules. 100 µL of isolated EVs contained approximately 5 × 10⁹ particles. Therefore, the labelling reactions were performed at a fluorophore excess of 4.8 × 10⁵. Considering that there are multiple available NH2 groups per EV particle, it is likely that at this ratio there was an excess of fluorophores over NH2 groups. To verify that all unbound fluorophores have been washed out to an extend that they do not affect cell uptake studies, a control containing only fluorophores but not particles was prepared. Therefore, the same amount of fluorophore was incubated with PBS and washed according to the staining protocol. Incubation of this sample with HCT 116 cells did not result in an intracellular uptake signal above cell background in flow cytometry measurements.

30 mL of conditioned medium (CM) was collected from approximately 2 × 10⁷ sub‐confluent HCT cells cultured in serum‐free medium for 24 h. CM was centrifuged at 3000 g at 4°C for 20 min. Supernatants were collected and concentrated 20‐fold using Amicon Ultra‐15 30 kDa MWCO centrifugal filters (Merck Millipore, Darmstadt, Germany). EVs were isolated by size exclusion chromatography (SEC) according to Brahmer et al. (2019 (link)). 10 mL syringes were packed with Sepharose CL‐2B (Thermo Fisher Scientific). Columns were washed with one column volume PBS before applying 2 mL of concentrated and precleared CM. Fractions of 1 mL were collected and analyzed for particle count by nanoparticle tracking analysis. Fractions 4–6 were collected as particle‐rich fractions and concentrated 30‐fold using Amicon Ultra‐2 30 kDa MWCO centrifugal filters (Merck Millipore, Darmstadt, Germany). Protein content was measured using Pierce 660 nm Protein Assay Reagent (Thermo Fisher Scientific). Absorption was measured at 660 nm with a M1000 plate reader (Tecan, Männedorf, Switzerland).