Exoquick tc kit

Exosomes were isolated from the culture medium of FHC, HCT116 and DLD1 cells by using an ExoQuick TC kit (SBI, USA), and exosomes from the plasma of subjects were purified by using an ExoQuick Plasma Prep with Thrombin kit (SBI, USA). The size distribution of the exosomes was characterized by nanoflow cytometry using a U30 Flow NanoAnalyzer (NanoFCM, Inc., China) with technical assistance provided by KeyGEN Biotech Co. Ltd. (Jiangsu Province, China). The shape and size of the exosomes were observed by transmission electron microscopy (TEM) (Tecnai G2, FEI, USA). Moreover, the characterization of the exosomes was confirmed by the presence of exosomal protein markers TSG101 (# ab125011, Abcam, USA) and Alix (# 92880, CST, USA). The green fluorescent dye PKH67 (Umibio, China) was utilized to label exosomes isolated from the culture medium of cells. After dye was incubated with recipient cells for 3 h, fluorescence microscopy (Zeiss, Germany) was performed to visualize PKH67-labelled exosomes in recipient cells. The detailed procedures were described in our previous study [27 (link)].

EVs were isolated via ASC-CM centrifugation at 4000 g for 30-min through a 100 kDa centrifuge filter (same as above) followed by washing with PBS and re-centrifugation at 4000 g for 5-min through the 100 kDa filter, for a total of 3x washes. EVs were precipitated from the remaining “ > 100 kDa” concentrate overnight using a ExoQuick-TC kit (SBI; Cat. # EXOTC10A-1), per the manufacturer’s protocol. EV were resuspended in PBS and aliquots were removed and used to quantify protein content as an indirect measure of EV content via QuickDrop, BCA, and Bradford (Coomassie). Relative EV protein fraction was compared to total protein within “ > 100 kDa” and “Full” ASC-CM fractions. Additionally, purified EV samples were then evaluated via Nanoparticle Tracking Analysis (NTA; Malvern Panalytical; Nanosight LM10) to further determine both concentration and size distribution of particles extracted from the ASC-CM, with exosomes typically ranging from 25 to 250 nm (n = 4). Unused (not exposed to cells) serum-free ASC media underwent the same processing and was used to establish/determine baseline EV/particulate levels, which were negligible. EV particle counts of serum-free control ASC media were negligible and did not warrant a full workup, thus only a comparative analysis between 2 and 3D was fully conducted.

At 70–80% cell confluence, the basal medium was replaced for cell culturing and the NF and CAF supernatants were collected after another 48 h. Cells were removed by centrifugation at 2000 rpm for 5 min, and cellular debris was removed using a 0.22-μm filter. Exosomes were then isolated from the supernatant using the Exoquick-TC kit (SBI, CA, USA). The obtained exosomes were resuspended in PBS and stored at − 80 °C after aliquotting. A bicinchoninic acid kit was used to determine the concentration of exosomes. To analyze the effect of CAF exosomes on MMECs, 2 µg of exosomes was added to 1 × 10⁵ MMECs for at least 48 h.