From: Albert Tai (acktai@exelixis.com)
Date: Tue Feb 18 2003 - 15:11:03 EST
Hi Cytometrists, Thanks for all the very valuable responses that give me suggestions on how to proceed with PI staining on ZsGreen expressing cells. This site is a great resource for all that involve in flow cytometry. Here is the responses that I received and would like to share with others that are interested. "It is possible that you are "loosing" the cells expressing low levels of GFP during your compensation as PI staining will be somewhat variable... You can try titrating down your PI staining and see if this corrects the problem." "To really get a good comparison, you need the same conditions with and without PI. That is, do you see the same 2-fold increase in apparent green fluorescence when you fix/permeabilize with 0.05% detergent when no PI is present? If no, then you may be seeing loss of fluorescent protein with permeabilization." "if ZsGreen is a form of GFP, you are probably denaturing the GFP with the detergent. the archives of this list has numerous discussions of how to perform cell-cycle analysis of GFP-expressing cells - you need to modify the protocol (including fixation) a bit to not damage the GFP." "soundsl ike simply that the PI is quenching the fluorescence. I would suggest using an alternative DNA like 7AAD which doesn't quench. If you have a UV laser then use hoechst" "you should only use 1 ug/ml of PI with fixed cells and I think its good to leave them O/N at 4oC for stoichiometric PI DNA binding. You willget less quenching thisway" Thanks very much again for your help! Albert Tai Exelixis, Inc. South San Francisco ----- Original Message ----- From: Albert Tai To: Cytometry Mailing List Sent: Friday, February 14, 2003 5:51 PM Subject: ZsGreen expressing cells with PI staining Hi Cytometrists, As a relatively new comer to 2-color flow cytometry, I have experienced some observation that I cannot explain and hope to get some insights from the experts in this forum. The experiment involves PI staining fixed cells that are transfected with ZsGreen vector expressing a transgene of interest. I am intersted in looking at the cell cycle profile of green cells (which expressed the transgene) vs non-green cells (control). As an indication of the transfection efficiency, I also trasfected the same cells with the same vector but did not stain the cells with PI (they were also fixed with 0.5% paraformaldehyde). The transfection efficiency of the fixed cells without PI was consistently higher than the ones that stained with PI, roughly 2X. For staining with PI, I also added detergent @ 0.05% as final conc. I would think if it is a compensation issue, it would be the other way. Any thought or comment is much appreciated. Hope you all have a nice weekend. Regards, Albert Tai Exelixis, Inc. South San Francisco, CA
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