Hi everyone, hoping that there might be someone who has done protoplast transfections who could help me out. For about six months now I've been doing transfections in Arabidopsis mesophyll protoplasts following the method described by the Sheen lab. The only difference between their protocol and mine is that my source of enzymes is from a different company.
I've had variable success. At one point I was getting 90% transfection efficiency, then it went down to about 50%, then nothing. I shortened the enzyme digestion time and my efficiency came back up. It worked well for several weeks, but now for about the past 6 weeks I get very few cells transfected, usually less than 1%. I think that it has something to do with the cells dying before I even get to the transfection step because if I stain them with propidium iodide, a stain which only stains dead cells, most all of the cells fluoresce. So I believe that it must be some step in the protoplast isolation procedure itself that is causing the cells to die, but I can't figure out what it is. I am doing nothing different that I can think of. I've tried shortening the digestion time down to 1-2 hours and using less of the enzymes. I've tried making all new solutions. There has been no improvement. I know that the quality of the plant material can be a factor, yet the plants look the same as they always have and I have transfected mutants plants that were practically dying without any trouble. This is frustrating me to the point of no return. Any help or suggestions would be much appreciated.
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