Real time analysis of microvesiculation using a Quartz Crystal Microbalance

Introduction: Characterization of microvesicles (MVs) is essential for understanding their mechanisms of action and biological importance. Stimulated MVs (sMVs) are released through the activation of cells by a multitude of factors. We aimed to use a quartz crystal microbalance (QCM) or piezoelectric quartz resonator able to determine small mass changes, to monitor MV release and to determine MV mass. Methods: A QCM (Q-Sence E1) was used to analyse MV release from THP-1 leukaemic promonocytes. The cells in RPMI and 2 mM Ca2_ were applied to the QCM to establish a steady baseline. The sample on the sensor was stimulated to microvesiculate with 10% exosome- and MV-free normal human serum. The QCM was then able to monitor sample density and fluid rigidity. Over the same time frame, the level of apoptosis of cells releasing MVs was assessed by staining with annexin V and 7-aminoactinomycin D (Guava Nexin Reagent). Using the QCM we were also able to measure MV mass directly by measuring their ability to quench the oscillating momentum of the QCM. Results: Using the QCM, we were able to monitor deposition of cells on the crystal and then sMV release from cells, in the absence of any labelling or fluorescent probe, by measuring cell mass change. Cells (105) were deposited onto the QCM electrodes, and the frequency decreases over the first 1000s indicating attachment. The cells were then stimulated with 10% EV-free NHS in RPMI and Ca2_ (2 mM) or, as a control, with heat inactivated NHS. During the ensuing 6.5 min, the resonate frequency remained stable. Then, over the following 10 min there was a 30 Hz increase indicating a loss in mass, consistent with the high rate of sMV observed. Given the crystal constant, C as 17.7, ^f as 19 Hz and v (the third overtone) as 3, and with the crystal area at 0.2 cm2, using the Sauerbrey equation we calculated the mass loss to be 23 ng which corresponded to 0.25 pg per MV given that 0.92 _ 105 MVs were released. The 16 min period over which MVs continue to be released as determined on the QCM coincides with the MV increase measured by FACS and with an increase in early apoptosis from 4% plateauing at 10%, levels of late apoptosis remaining at 1_3%. We also looked at deposition of sMV on the sensor. Given a Df of 27197 Hz for the deposition of 1.3_106 sMVs, we estimate the mass of an sMV by this approach as 0.24190.006 pg. Summary/conclusion: Using the QCM we were able to measure a significant change in cellular mass, beginning at 6.5 min post-stimulus and peaking at 1000 s post-stimulus. The QCM also detected a decrease in media fluidity, attributed to the process of membrane blebbing on THP-1 and MV release. The QCM was able to provide an accurate measurement of sMV mass (0.25 pg) by calculating the loss in mass of the stimulated cells. By measuring the quenching of the oscillating momentum on the QCM as sMVs are deposited on the sensor, we were also able to calculate the mass of an sMV as 0.24 pg.