Gene delivery into cells via microfluidic device
To examine the potential of the fabricated design to enable intracellular delivery, target cells were centrifuged at 1200 rpm for 5 minutes. Cell pellets were dissolved in 5% serum containing medium. Myeloma cells and plasmids were introduced into the microfluidic transfection device either in tandem or sequentially by flowing suspension medium. Various velocities were investigated to generate chaotic advection in the device. The speed was set at a range of 3-13 ml / hr. After completion of process, the cells were Collected and suspended in appropriate culture media and plated in appropriately sized standard culture plates at 2 × 106 cells/ml which incubated in a 37 ° C incubator with 5% CO2.
Flow CytometryTo analyze the percentage of GFP positive cells by BD FACS Calibur Flow Cytometer, PE-anti BCMA antibody (407107) was purchased from Biolegend. Cells for flow analysis were suspended in flow buffer (PBS with 0.5% BSA) containing antibody and incubated for 15 min at room temperature.
Cells were washed with PBS, re suspended in 500μl PBS- BSA1% and 10 μg/ml propidium iodide (PI) was added immediately before flow cytometry analysis. The transfection efficiency was determined 24 - 48 hours post transfection.
Statistical AnalysisStatistical analysis was performed using GraphPad Prism 6 and FlowJo. All data are represented as means ± SD.