I am trying to streamline a nuclear fractionation from liver tissue and was having great luck using a nylon mesh strainer to grind the tissue into a single cell suspension before beginning.  Someone recommended a dounce homogenizer to save cost.  I have never used such a relic lol.  I have tried a few samples but something isn't right by the time I get to the cytosolic fraction it seems as though my nuclei are lysing and I am only left with a huge DNA pellet.  I have read a ton of protocols and commonly see that if the dounce homogenizer is used with lysis buffer to yield nuclei upon homogenization.  My question is, will using only PBS create a single cell suspension that I can then use with my subcellular fractionation protocol or am I lysing the cells by a force unbeknownst to me?  I have looked at the nuclei using trypan and am not seeing as many nuclei as expected in the nuclear fraction.  Is it possible that the force of the homogenizer from pestle A to B is lysing the cells?  In general this is the steps I am taking, I can't get scoped so it is the quick version.

1. Create single cell suspension from liver tissue (~50mg)

a. use nylon mesh strainer and gentle grinding with PBS 

b. use dounce homogenizer with PBS 

2. Spin low speed to pellet "intact" cells (think media change spin)

3. Resuspend in 0.1% NP-40/PBS - mix pellet and take aliquot for whole cell lysis 

4. Spin - remove supernatant (cytosolic fraction), remaining pellet should be nuclear.   

Thank you so much for your help!