From: Sara Turner (sturner@zoo.uvm.edu)
Date: Wed Nov 18 1998 - 09:17:15 EST
I've been trying to use EGFP-F as a transfection marker, and having problems with *all* of the GFP transfected cells (2 peaks) shifting significantly to the right of the empty vector transfected cells on the green histogram. I've done a couple of experiments to try to figure out what to do about that, and I thought I'd share my results. -- If I mix EtOH fixed GFP transfected samples with untransfected, fixed cells I see three distinct peaks on the histogram. -- If I mix the live cells before the EtOH fix step, I see the entire population (2 peaks) migrating to the right on the histogram. The longer the cells are together, the farther to the right the peaks shift. A significant shift was seen even when the cells were together for a few minutes before the EtOH was added. I also tried a bunch of sample prep conditions... -- Rinsing the plate of cells a couple of times with PBS the day after transfection and then feeding the cells with fresh media resulted in a slight left-ward shift in the 2 green peaks compared to the unrinsed but fed cells. -- Washing with PBS/1% BSA after EtOH fixation also resulted in a small leftward shift compared to the cells washed with plain PBS. -- Overnight EtOH fixation resulted in two nice, distinct peaks for the - and + GFP cells (EGFP-F). Fixing for 30 minutes was not bad, but there was less of a dip between the two peaks. The short fix time would probably be sufficient in a pinch, but the O/N fix looked prettier. -- There was no difference if I washed the cells with PBS/1% BSA or plain PBS before the EtOH fix step. -- The EGFP-spectrin peaks did not appear as far to the right as the EGFP-F peaks, but there didn't seem to be as distinct of a + peak as with the EGFP-F. The spectrin had more of a "shoulder" than a peak. All of the above improvements were relatively subtle. Both the + and - peaks for the GFP (spectrin and F) transfected cells still appeared well to the right of the empty vector transfected cells. I think decreasing the amount of plasmid may improve that problem a bit, and I'll still be able to see a definate + population. I'll work on tweeking the plasmid amount again. Maybe that will help. Sara Turner Another flow newbie. >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 > _____________________________ _____________________________ Sara Turner University of Vermont Medical School Pathology Department (Dr. James Koh's Lab) Medical Alumni Building, room A221 Burlington, VT 05405 sturner@zoo.uvm.edu
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