Hi there, I've got another problem for you all... I'm trying to isolate nuclei from CFSE stained cells so that I can then fix and permeabilize the nuclei, perform intranuclear staining, and analyse using flow cytometry. I am having great trouble with finding a suitable lysis protocol; after the initial incubation and centrifugation step, I am unable to recover many "usable" nuclei. Most of the nuclei form a large aggregate which can't be broken up by vortexing or using a pipette. To lyse my cells, I've been using HBSS/0.1%BSA/0.1%NP- 40 for 5 mins, or 1%NP-40 in 20mM EDTA, 50mM Tris-HCl pH7.5 for 10 seconds. I have included NP-40 in the wash buffers, in concentrations ranging from 0.02% to 0.1%, and I have varied the centrifugation speed from 1600rpm to 8000rpm. The actual lysis step is fine, and I assume the problem is due to the DNA being sticky. Obviously these clumps are bad for running on the flow and I've thought about including RNAse in the lysis and wash solutions but haven't got around to trying this yet. Has anyone else tried to analyse isolated nuclei using flow cytometry, or does anyone know a way around this problem? Thanks, Andrea Andrea Dewar PhD Student Leukaemia Research Laboratory Institute of Medical and Veterinary Science Adelaide South Australia AUSTRALIA 5000
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