Fig. 5.

Analysis of the TF antigen content and fluorescence of TF-tGFP-containing microvesicle fractions separated by density gradient centrifugation. MDA-MB-231 cells were transfected to express the TF-tGFP hybrid protein. Cells were washed and pre-adapted to respective serum-free media prior to activation with PAR2-AP (SLIGRL; 20 µM). The conditioned media were collected and cleared of any cell debris by centrifuging at 5,400g for 10 min on a microcentrifuge. Cell-derived microvesicles were then sedimented at 20,000g and 100,000g at 20°C for 1 h. Microvesicles were analysed by density gradient ultracentrifugation using a sucrose–OptiPrep gradient covering an approximate range of 1.02–1.22 g/ml against 2 sets of DensityMarkerBeads as density markers. The samples and markers were centrifuged at 52,000g for 90 min at 20°C. Following centrifugation, aliquots (0.5 ml) were sequentially removed. The TF antigen content of the samples was determined using the TF-EIA procedure (A). The presence of TF-tGFP protein was assessed by measuring the fluorescence at 510 nm and subtracting the values obtained from a similar set of microvesicles but from untransfected cells (B) (n=3).