Dear Chiara Cavallini,

In order to answer your question, could you provide some more information? Can you specify which flowcytometer, which type of lightsources and detectors are at your disposal? PI is not the most suitable stain for obtaining low CV histograms, DAPI is generally considered to be a better choice, but it requires excitation at 360 nm and detection in the low 400 nm range. But with some machines, CV's of 1 % or lower can be obtained. The most simple protocol, starting with protoplasts, is just adding a staining buffer, with 0.1% Triton -X100, Dapi at 1 ug/ml and allow to stand for 15 min and measure. For leaf material, just use a razor blade (new, sharp) and just cut some leaves in a suitable buffer with the same composition. Filter through 50 um mesh nylon filter gauze and measure. The same procedure can be done with PI at 10 ug/ml, but with higher CV's. Kind regards, Harrie Verhoeven.

Dear Harrie Verhoeven,

thank you very much for the kind response. We will use a FACSCanto (Beckton Dickinson) flow cytometer equipped with blu (488 nm) and red (635 nm) lasers. the opltical filter are for the blu laser: 488/10 (SSC), 530/30 (FITC), 585/42 (PE), 670LP (PerCP), 780/60 (PECy7) and for the red laser: 660/20 (APC), 780/60 (APCCy7). We have decided to try the staining with PI because we routinely use it for the cell cycle in human cells and the flow cytometer can read it. Thank you again for your help, best regards, Chiara