Dear Group, re: Superfect negative controls and autofluorescence I have been asked to look at eGFP expression in PBMCs that have transfected using a cationic lipid carrier (Superfect from Qiagen). The cells are then cultured for 24 hours, harvsted with EDTA or scraping, blocked in 10% goat serum/PBS, stained for subtypes with PE conjugates, and finally resuspended in 1% PFA and run the following day. I am using a FACS Caliber with the stock FL1 filter 530-30 replaced with a 530-40 with the idea being to get closer to the emmission max of eGFP (507nm? 510nm?) For a negative control I have cells that been exposed to just the Superfect (no DNA) and see large amounts of background fluorescence. This is also seen under the scope (535-45 filter). It would be great to hear if people have done flow on cells after similar exposure. Do I have the most appropriate negative controls? Does superfect autofluoresce? If so have people dimmed this with anything? I also have a question into the supplier and am waiting for their response. Any great references to point out? Thanks for any imput, Peter Schroeder Scientist Materials and Bioscience Center Medtronic, Inc. Minneapolis, MN
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