4.6. Flow Cytometric Analysis
The expression of sGFP was monitored at 24 h following transfection using a FACS Aria II flow cytometry system (BD). Protoplasts were pelleted and resuspended in an appropriate volume of PBS buffer and immediately subjected to flow cytometry. Flow cytometry analysis was performed as described by Hagenbeek and Rock [9]. The cell density and sample injection speed was adjusted to the particular experiment for the best possible yield or fastest achievable speed. Live protoplasts were gated using the forward and side scattering fields for data acquisition; irregular shaped protoplasts were regarded as dead or aggregated and were gated out during the data collection. As shown in Figure 6, non-single cells, such as cell-multiplets or cell debris, were excluded through a large initial gating of a side scattering vs. a forward scattering (FSC)-A, and two more gating steps of a SSC-height vs. -width and a FSC-height vs. -width were used more strictly to exclude non-single cells from the next analysis. The green fluorescence of sGFP was excited by a 488 nm laser and detected using the FITC-A channel. For each analysis, the sGFP fluorescence signals of 105 events in the gated population were collected and detected using the FITC-A channel and analyzed using FACS Diva software 6.0 (BD). Control protoplasts were prepared through PEG transfection with no plasmid as a negative control. The fluorescence intensities of the control samples (100–103), derived from chlorophyll in the chloroplasts, provided the autofluorescence background value for FCA of other protoplasts expressing sGFP proteins.
Figure 6  The scatter plots of representative data from FCA of rice protoplasts. The protoplast populations were initially gated through a forward scatter (FSC) area versus a side scatter (SSC) area to exclude non-single cells such as cell-multiplets or cell debris. Afterwards, further gates were performed using a SSC-height vs. SSC-width dot plot and a FSC-height vs. FSC-width to more strictly select single protoplast cells.