I am doing this experiment now: agrobacteria carry a viral vector with a EGFP signal, and I tried to figure out the ratio of cells that are infected (have EGFP).The plant I used is N. benthamiana. so I collected the cells, did a protoplast prep to digest cell walls and sent the cells to cytometer. the major issue I have : in the first optimization step (adjust volts for FSC vs SSC), I cannot get the dots to move to the center. most of time they form a sector. I tried to fool around with different volts/amps setting but the best I can do is to make most dots about the same FSC around 200 and SSC always vary (Fig 2). Needless to say, further steps looked ugly because there is no way I can gate the cells I actually need (or separate debris).
Wonder if it's actually doable to run epidermal cells on cytometer? or any suggestions on what to do to solve the issue?
- Badges
- Science method