It’s never too late to take another look at your data.
The incredibly knowledgeable Sally Cowley suggested that I shouldn’t just think about how many cells contain any AlexFluor488 antibody after electroporation, but how bright each cell is (i.e. the amplitude of fluorescence). Looking at this instead will give me an idea of how much protein enters each cell. So. I went back to the old data, saw that the electroporation pre-sets do have an effect on fluorescence amplitude, and added a new gate to quantify the difference.
With this I could see that my previous best pre-set produced only 27% “hyper-positive” cells, compared with 93% hyper-positive cells in the new Best Electroporation Pre-set!
At the same time I can see that the three best pre-sets also produce the highest levels of cell death, but I think this is actually a good sign. If the electroporation has more of an impact on the cell membrane it makes sense that more protein can enter the cell, but also that more of them will die. And the level of cell death is still low enough that in the end there are more hyper-positive cells.
Hopefully this rethink will make my future editing attempts even more effective!
You can read the full details of what I did in my Zenodo report.