Bill, I do not think you need to use ETOH if you have fixed your cells with PFA for membrane staining and PI cell cycle. After surface staining and fixation resuspend the cells in 0.1% Na citrate, 0.1% Triton X-100, 50 ug/ml PI and eventually RNAse 50 ug/ml. Leave cells ON and then analyse by FACS. That will make the job. Remember that with PFA fixation PI profiles do not look as good as if you use ETOH to fix. On the other hand surface staining is in most cases affected by ETOH fixation. You have to find the compromise which best fit your purpose. Hope it helps Corrado Corrado M. Cilio, M.D., Ph.D. Immunology Research Unit Dept. of Endocrinology Malmo University Hospital Lund University, Malmo Sweden
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