Chapter 3 68 Framework Criteria What to report Please complete each criterion 2.1 Sample staining details State any steps relating to the staining of samples. Along with the method used for staining, provide relevant reagent descriptions as listed in MIFlowCyt guidelines (Section 2.4 Fluorescence Reagent(s) Descriptions). Sample staining: 30 uL of sample was added to a pre-defined volume of fPBS (dependant on the volume of mAb staining - total volume after mAb addition was set at 130 µL): 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: anti-CD9 – 2.5 ng, anti-CD63 – 83 ng, anti-CD81 – 83 ng, anti-CD31 (anti-human) – 1 ng, anti-CD31 (anti-mouse) – 40 ng per test. Equivalent amounts of isotype control was used for each antibody. Samples were then incubated overnight at 4 °C to ensure optimal saturation of the available EV epitopes; this incubation time was determined 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 of incubation at room temperature in the dark. Control samples not stained with CFDA-SE were incubated with 100 µL fPBS instead. All samples were brought to a total volume of 380 µL using fPBS before IFCM measurements. 2.2 Sample washing details State any steps relating to the washing of samples. No sample washing was performed; background fluorescence induced by our protocol is described in detail in this work. 2.3 Sample dilution details All methods and steps relating to sample dilution. For sample staining (descibed above), 30 uL of sample was incubated O/N in a total volume of 130 uL, resuling in 4.25-fold sample dilution. This volume was topped-up with 250 uL fPBS to a total volume of 380 uL, resulting in a total dilution of ~12.6-fold. For serial dilution experiments, samples were diluted four times (4-fold each step) by mixing 100 uL of sample with 300 uL of fPBS.
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