Making Nuclei From Mouse Cells - (Mar/08/2012 )
I need help making nuclei from mouse bone marrow cells. My end goal is to use DNase and look at hypersensitivity regions for genes of interest and therefore my nuclei preparation is critical to the success of the assay. The basic problem I have is that I'm completely unable to count the nuclei.
I'm currently using 0.5% NP-40 in a 4 ml volume of buffer that includes 15mM Tris-HCl, 15 mM NaCl, 60mM KCl, 1mM EDTA, 0.5 mM EGTA. I incubate 20 million cells in this NP-40 based buffer for 10 minutes on ice, then spin for 1000 rpm, 5 min.
After the spin, I attempt to resuspend the cells in buffer without NP-40. However, it is at this point where I'm not able to resuspend the nuclei. They stick together in a clump. I'd like to count them, but of course cannot do so accurately.
Has anyone had this experience in making nuclei? Do you even bother to count them or just proceed with your protocol? Are there any ways to resuspend the nuclei and break them apart so they can be viewed under a microscope?
The stickiness is probably due to some of the nuclei lysing, but in my experience the number of cells you have per ml is too low for that to be happening. I would check the buffer components (especially the salt concentration, 15 mM is probably too low, it should be around 150 mM for normal saline), and the lysis conditions, 10 min seems short to me.