Hi, Nagesh, you can try other dye, for example DAPI. Moreover, DAPI is more specific for DNA, so, you may easy determine DNA: propidim may alos bind to RNA. I think debris should not stain with DAPI. Good luck!

Agree with Taras's comment. First try with DAPI to see if that improves things. Thereafter, you can try diminishing the pore size of the filters used or simply let samples decant for a few minutes in the fridge before measurements.

Hello, you can try a iodixanol gradient to purify the nuclei with this protocole : http://www.axis-shield-density-gradient-media.com/training-6new.ppt

or you can separate the debris from the nuclei in relation to their size. This is possible when you do your cytometry analysis. Good luck, Isabelle

I am not in disagreement with the previous comments about the DAPI, but in my opinion the PI is more accurate than the DAPI to quantify the genome size of the nuclei by flow cytometry. I am using the PI to estimate the genome size in vine cacti, which is a very difficult specie to quantify the 2C DNA nuclei content , due to the high polysaccharides content in the young stem tips. Followed you can find my methodology and I hope that it will be helpful for you:

FACS analysis and ploidy estimation

Nuclear suspensions were prepared by chopping 60-100 mg of tissue from the young shoot tips of the stems in a petri dish with a razor blade in 1 ml of ice-cold nuclei isolation buffer (NIB) prepared according to Doležel et al. (1989), to which 1 µl/ml β mercaptoethanol was added immediately before use. To obtain acceptable coefficient of variation values, 0.1% Triton® X–100 for reducing the adhesion of cellular debris and 5% polyvinylpirrolidone PVP–40 for binding phenolic compounds, were added to the NIB. After chopping, the samples were transferred to a 15-ml polypropylene tube, 2 ml NIB were added, and the tube was placed on the shaker for 1 h. After shaking, the samples were filtered through a 50 µm nylon mesh (to remove chopped tissues, cell fragments, and large debris) and concentrated by centrifugation at 1900 rpm for 8 min. The supernatant was discarded and the concentrated nuclei were re-suspended with 1 ml ice-cold staining NIB supplemented with 50 µg/ml of DNase-free RNase A and propidium iodide. To facilitate flow cytometric recording, the samples were filtered again through a 35-µm mesh (Falcon 12×75 mm tubes with a 35-µm strainer cap). The ploidy of regenerated plantlets was determined by comparing their G0/G1 peak positions with those of the diploid H. monacanthus and the tetraploid H. megalanthus used as external standards, both of which were previously standardized against the internal standard the Pisum sativum cv. Ctirad as a genome size reference (2C = 9.09 pg of DNA).

I would suggest you to try out Otto's buffer. This can earn you a good result. 

Otto I buffer (Otto, 1990): 100 mM Citric Acid; 0.5 % (v/v) Tween 20 (pH approx. 2.3)

Otto II buffer (Doležel and Göhde, 1995): 400 mM Na 2 HPO 4 .12H 2 O (pH approx 8.9) 

Take around 50 mg leaf tissue without midrib, chop it in 500 ul of Otto I buffer. Add 100 ug/ ml conc of PI in 500 ul Otto II buffer. Mix it with 500 ul of nuclei suspension in Otto I. Pass the nuclei suspension through a 50 micron nylon mesh, run in FCM.