Exosomal biomarkers measurement
tumor, ABM
TUMOR, LYSIS, SECONDARY
The total protein content of exosomes was extracted using a homemade cell lysis buffer, followed by protein precipitation using a 5 × protein loading buffer (ABM, Vancouver, Canada). After 5 min of heating in a water bath at 100 °C, the mixture was transferred to a polyvinylidene difluoride membrane (Millipore, Burlington, MA, USA). The PVDF membrane was blocked by incubating it for 1 h at room temperature in milk Tris-buffered saline with Tween20 solution. After that, the membrane was incubated for 15 h with primary antibodies against rabbit anti-mouse CD63 (1:1000; Abcam, USA) and tumor susceptibility gene 101 (TSG101; 1:1000; Abcam, USA). The membranes were then incubated for 1 h at room temperature with HRP-conjugated secondary antibodies (1:1500; Abcam, USA). Finally, the expression of proteins was determined using enhanced chemiluminescence reagents.