Journal: Journal of Extracellular Vesicles

Article Title: Characterization of physical properties of tissue factor–containing microvesicles and a comparison of ultracentrifuge-based recovery procedures

doi: 10.3402/jev.v3.23592

Figure Lengend Snippet: Nanoparticle tracking analysis (NTA) of TF-containing microvesicles (1.03–1.08 g/ml) separated by density gradient centrifugation. Microvesicles were prepared from normal human plasma (A), conditioned media from MDA-MB-231 cells (B), and conditioned media from MDA-MB-231 cells expressing TF-tGFP protein (C). The plasma and conditioned media were collected and cleared of any cell debris by centrifuging at 5,400 g on a microcentrifuge, and microvesicles sedimented at 100,000 g . The microvesicles were resuspended in PBS and were fractionated by density gradient ultracentrifugation using a sucrose–OptiPrep gradient covering an approximate density range of 1.02–1.22 g/ml alongside 2 sets of DensityMarkerBeads. The samples were centrifuged at 52,000 g for 90 min at 20°C in a SW41Ti rotor on a Beckman L8-M ultracentrifuge. Following centrifugation, aliquots (0.5 ml) were sequentially removed and assessed for TF antigen. Samples containing TF antigen were then pooled (1.03–1.08 g/ml) and diluted 1:10 in PBS, and the size of the microvesicle population was analysed by NTA using a NanoSight LM10 instrument. A control sample was prepared by adding MDA-MB-231-derived microvesicles to the same pooled fractions from a blank density gradient centrifugation (D). A negative control made of the pooled fractions from a blank density gradient centrifugation showed no detectable trace (E). The illustrations are typical (n=3) of the size distributions which were determined using NTA software. The total amounts of microvesicles in the samples are not comparable. TF-containing microvesicles were immuno-purified from conditioned media of MDA-MB-231 (F) and A375 cell lines (G). The samples were incubated with a monoclonal antibody against TF (10H10; 4 µg/ml) followed by protein A-magnetic beads. The samples were washed with PBS and eluted in phosphate buffer containing NaCl (500 mM). The samples were analysed by NTA against a sample treated similarly but without the antibody.

Article Snippet: TF-containing microvesicles were also isolated directly from conditioned media of MDA-MB-231 and A375 cell lines, by immuno-precipitation using a monoclonal antibody against TF (10H10) (Santa Cruz Biotechnology, Heidelberg, Germany), followed by protein A magnetic beads (Miltenyi Biotec, Bisley, UK).

Techniques: Gradient Centrifugation, Clinical Proteomics, Expressing, Centrifugation, Control, Derivative Assay, Negative Control, Software, Purification, Incubation, Magnetic Beads