The effect of centrifuge speed and the amount of microbubbble
solution on the delivery efficiency were separately identified and
quantified by flow cytometry. Fig. 3a show the result of delivery efficiency at different centrifuge speeds: different centrifuge speeds
have no significant impact on delivery efficiency. The delivery efficiency could reach 90% even when the centrifuge speed is only
1g (100 RPM). Similarly, the delivery efficiencies were independent of the amount of microbubble solution, as shown in Fig. 3b.
The efficiencies are around 85–90%. The FACS histogramsof cell fluorescence are shown in Fig. 3c and d. The horizontalaxis is the fluorescence strength, while the vertical axis stands for the cell number at
certain fluorescence strength. On Fig. 3c protoplasts centrifuged
alone are shown as a single population with low fluorescent background on the left. The peak of the fluorescence distribution of protoplasts mixed with FITC for 6 min (blue curve) shifted to the right.
The fluorescence distribution of protoplasts centrifuged with microbubbles at 1g (100 RPM) (green curve) shows two peaks and
shifted to the far right. This phenomenon shows that after the microbubble assisted centrifugation process, the protoplasts have a stronger fluorescence signal, which also means the microbubbles assisted
centrifugation process did help improve the delivery efficiency. To
clearly present the delivery difference between samples centrifuged
with different amounts of microbubble solution, the FACS histograms were compared between samples centrifuged with 5 and
65 lL of microbubble solution as shown in Fig. 3d. The fluorescence
distribution for canola protoplasts centrifuged with 65 lL of
microubble solution was much sharper than that with 5 lL of microbubble solution. In other words, more canola protoplasts have uptaken a similar amount of FITC in the sample centrifuged with
65 lL of microbubble solution. The reasons might be that the extra
amount of microbubbles opened extra pores on the cell membrane
of one single protoplast, or a greater population of overall protoplasts increases diffusing FITC molecules into the cells. Fig. 3e and f
show the confocal microscopy images of canola protoplasts centrifuged alone and canola protoplasts with FITC delivered by microbubbles assisted centrifugation. The blue color stands for the cell
nucleus, while the green fluorescent color was emitted by FITC