To begin, take Calu6 cells transfected with fluorophore-conjugated EV-microRNA and cell-microRNA. Filter the cell suspension through a 35 micrometer strainer cap included with the round bottom polystyrene FACS tubes. Then set up the flow cytometry instrument, switch on the flow cytometer, and allow it to warm up for 30 minutes before use.
Prime the fluidic system with sheath fluid. After that, verify and adjust the laser alignment. For quality control, load ultrapure filtered water into the flow cytometer and run it at an appropriate flow rate, ensuring an adequate acquisition time.
Then set the threshold for capturing the signal from Calu6 cells at FSC 5, 000. To account for spectral overlap between fluorophores, prepare compensation controls using untransfected cells and cells transfected with only one fluorophore. Perform expression analysis in transfected cells using the FSC, SSC, FITC, and PE channels set at 258, 192, 269, and 423 voltage units respectively.
Then introduce the negative control sample into the flow cytometer. Capture cell signals after defining FSC and SSC intensities in a custom worksheet. Select FSC for the X-axis and SSC for the Y axis in the plot.
Draw a polygon around the population of interest to create a gate on the axis. To create a new plot using the gated population, set the X-axis to fluorescent signal 1. For cell-microRNA detection and the Y-axis to fluorescent signal 2 for EV-microRNA detection.
Establish compensation parameters for the individual fluorophores using cells independently transfected with each of the fluorescently tagged microRNAs. Flow cytometry analysis of cells transfected with cell-microRNA and EV-microRNA revealed a sequential decrease of fluorescence signal corresponding to EV-microRNA. In contrast, the signal corresponding to the cell-microRNA was retained in the cells, indicating that the fluorophore does not impact the retention of microRNAs by the cells.
