Calcium ion concentration from 0 mM to 0.25 mM resulted in a substantial (p0.001) boost in transfection efficiency (7.three ?0.eight vs. 22.7 ?0.five ) (figure 10a), fluorescence intensity (1.four ?106 ?0.1 ?106 RFU vs. 7.7 ?106 ?0.5 ?106 RFU) (figure 10b) as well as a important boost in cell viability (36.0 ?three.4 vs. 67.two ?1.7 ) (figure 10c). Nonetheless, upon increasing the calcium ion concentration additional no added enhance in transfection was achieved and between 1 and 1.5 mM there was a considerable (p0.001) drop in transfection (21.0 ?0.7 vs. 14.0 ?0.four ) which dropped to 11.9 ?0.6 at two mM. Cell viability, alternatively, remained continual at about 67 . Zhou et al. have shown that the time for comprehensive membrane resealing of sonoporated Xenopus oocytes is dependent on Ca2+ concentration. Complete resealing took 58?70 seconds with 0.54 mM Ca2+ but only six?six seconds at 1.8 mM Ca2+(Zhou et al. 2008). The boost in transfection observed in figure ten because the Ca2+ concentration decreased from two.0 to 0.25 mM might have been brought on by the cell membranes of damaged cells sealing much less rapidly when exposed to lower calcium ion concentrations allowing a lot more time for plasmid DNA to enter the cell. However, the drop in transfection efficiency and viability observed when the Ca2+ concentration was decreased to 0 mM confirms the observation that calcium is necessary for sonoporation which has been reported by other people (Schlicher et al. 2006). The calcium ion concentration within the blood is generally higher than two.1 mM and just isn’t very easily changed which may limit the transfection efficiency in some in vivo applications. In other applications including transfection with the salivary gland, exactly where the microbubble and plasmid suspension is often delivered into the lumen inside a controlled buffer, it might be valuable to optimize the calcium ion concentration. Effect of magnesium concentration Furthermore to requiring calcium, Steinhardt et al. have shown that resealing of disrupted cell membranes could be antagonized by magnesium ions (Steinhardt et al. 1994). This would recommend that larger concentrations of Mg2+ could slow the process of membrane resealing and permit extra plasmid to enter the cell. Having said that, as shown in figure 11, when Mg2+ was added collectively with 1 mM CaCl2 below exactly the same insonation situations as above, no substantial difference in transfection was observed for any with the Mg2+ concentrations tested (19.9 ?1.1 , 21.0 ?0.7 and 21.two ?0.eight for Mg2+ concentrations of 0, 0.5 and 1 mM, p0.05) (figure 11a). There was also no considerable transform in fluorescence intensity (six.Formula of 5-Bromo-3-nitropyridine-2-carbaldehyde 7 ?106 ?0.2,2′-Dibromo-1,1′-biphenyl web 3 ?106 RFU, 7.PMID:25147652 0 ?106 ?0.four ?106 RFU and 9.0 ?106 ?0.5 ?106 RFU) (figure 11b) or cell viability (71.4 ?1.4 , 66.5 ?two.three and 65.6 ?2.7 for 0, 0.five and 1 mM, p0.05) (figure 11c). Many exposures As shown in figure eight, following two seconds of exposure no additional transfection was accomplished. This could possibly be resulting from a lack of adequate intact microbubbles soon after ultrasound exposure preventing addition sonoporation, or as a consequence of heterogeneity in the cell population creating some cells much less susceptible to transfection than other folks. Cells grown in tissue culture areUltrasound Med Biol. Author manuscript; readily available in PMC 2014 June 01.NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author ManuscriptCochran and WheatleyPageparticularly vulnerable to stressful conditions, and efforts to execute repeated transfection within a brief time frame (minutes) resulted in unacceptable loss of cells because of detachme.