From: Maggie Kasten (kasten1@jeflin.tju.edu)
Date: Fri Oct 30 1998 - 11:04:21 EST
Dear Flow-ers, This is a follow up post to one I made a month ago about background obtained with the EGFP-F plasmid. Several people expressed interest in this construct and so I hope this information is useful to them and others. Here's a review of our problem. EGFP-F is an EGFP construct targeted to the cell membrane through the ras farnesylation sequence and, therefore, should not leak out of ethanol fixed cells. However, flow cytometry analysis of our ethanol fixed cells transfected with EGFP-F showed significant increase in the fluorescence signal of the untransfected population. The increase was so severe that we despaired of using this construct in our flow cytometry experiments. Andy Beavis posted that he has seen similar results with EGFP-F. He was also kind enough to recommend a different membrane targeted EGFP construct, EGFP-Spectrin, that I received. In our hands EGFP-Spectrin also showed increased background, although not nearly as severe as EGFP-F. (Sorry Andy.) I received an e-mail message from a Clontech research scientist about the background problem. My thanks to the person who forwarded her my cry for help. She said that Clontech had heard that some researchers experience increased background when using EGFP-F with ethanol fixed cells. She believes that the background is due to "short" ethanol fixation and that the problem can be remedied by fixing cells for at least 24 hr or by washing the fixed cells with PBS + 1% BSA. I'm a bit skeptical about the short fixation explanation as we routinely fix our cells overnight. However, washing the cells with PBS + 1% BSA did eliminate our background while maintaining the brightly fluorescent cells. This addition worked to reduce the background for both the EGFP-F and the EGFP-Spectrin transfected cells. It seems unlikely that the BSA step will interfere with subsequent treatment of the cells, PI staining for cell cycle analysis in our case, and so should be generally useful. Thanks to all who responded. Maggie Kasten Flow Cytometry Newbie -- Maggie Kasten Voice: 215-503-4614 Pathology Department FAX: 215-923-9626 Thomas Jefferson University Philadelphia, PA
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