Detection of apoptosis in a microfluidic circulatory system

A microfluidic circulatory system (Figure ​(Figure3A)3A) was assembled by using a peristaltic pump (Ismatec, Germany) to generate a pulsatile flow in a circulatory silicone tubing (Ismatec, Germany) with a diameter of 500 μm and a total length of 1.5 m. A polydimethylsiloxane (PDMS)-based observation chip was linked with the microfluidic circulatory system when live cell imaging microscopy was conducted. SS in the tubing was calculated using Poiseuille's equation [4], τ = 4Qη/πR³, where τ is SS in dyne/cm², Q is flow rate in cm³/s, η is the dynamic viscosity of the fluid (the culture medium can be treated as water at 37°C; η = 0.01 dyne*s/cm²), and R is the radius of the silicone tubing (250 μm). The flow rate can be adjusted to achieve SS range from 5 to 30 dyne/cm². To prevent cells attaching to the tubing or the PDMS chip, the whole system was pre-coated with 0.5% Pluronic F127 in PBS (Invitrogen, USA) for 1 hour at room temperature before the experiments. A Pluronic F127-coated, 96-well plate was also used as a negative control for the no SS condition.

Cancer cells were collected using 0.05% trypsin containing 0.53 mM EDTA (Gibco, USA), washed twice with PBS and re-suspended in fresh culture medium at a cell density of 2 × 10⁵/ml before injected into the microfluidic system. One milliliter of cell suspension was added to the microfluidic circulatory system and subjected to circulation for varying times at 37°C in a humidified CO₂ incubator. For live cell imaging, the pump was switched off to stop the medium flow, and the movement of sensor cells was retained within the observation chip by closing the two control valves. The rate of apoptosis was determined by FRET imaging microscopy.