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.