Chapter 3 56 concentration: 3.2 µg/mL), respectively. The anti-tetraspanin antibody mixture was made by combining anti-CD9/anti-CD63/anti-CD81 in the same stock solution. Preparation of a carboxyfluorescein diacetate succinimidyl ester stock solution (Step V) A carboxyfluorescein diacetate succinimidyl ester (CFDA-SE) stock solution was made with the Vybrant™ CFDA-SE Cell Tracer Kit from Invitrogen immediately prior to use according to the manufacturer’s instructions. In brief, CFDA-SE powder was spun down using a table-top centrifuge, and 18 µL of dimethylsulfoxide (DMSO) was added. The mixture was thoroughly resuspended and incubated at room temperature for 10 – 15 minutes in the dark. Then, the dissolved CFDASE was added to a total volume of 1.782 mL of fPBS to create a 50 µM CFDA-SE stock solution. Similar to the protocol used to prepare mAbs, this stock solution was centrifuged for 10 minutes at 16,000 x g to reduce potential CFDA-SE clumps (Figure 9 – step V); the top layer was carefully harvested – leaving ~100 µL of liquid in the tube – before being added to the samples. Sample labeling (Step VI) Staining was performed overnight at 4 °C in the dark in a total volume of 130 µL. This volume was build-up by 30 µL of sample, a volume of mAb stock solutions (described under step IV) as needed and brought to the total volume of 130 µL with fPBS; 12.5 uL of the stock solutions containing mAbs labelled with –APC and 5 µL of the stock solutions containing mAbs labeled with –BV421 were added, resulting in the following concentrations used per test: CD9 – 2.5 ng, CD63 – 83 ng, CD81 – 83 ng, CD31 (anti-human) – 1 ng, CD31 (anti-mouse) – 40 ng per test. Equivalent amounts of isotype control were used for each antibody. For specificity and sensitivity analysis, human and mouse PPP were mixed at varying ratios with the total volume of PPP maintained at 30 µL. Samples were then incubated overnight at 4 °C to ensure optimal saturation of the available EV epitopes (Figure 9– step VI); this incubation time was determined empirically by adding the anti-tetraspanin antibody mix to fPBS and PPP samples and performing acquisition at set intervals (1/3/6 hours and O/N). CFDA-SE labeling was performed on the day of data acquisition by adding 100 µL of the 50 µM CFDA-SE stock solution to the samples, followed by 30 minutes
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