I am trying to use FC to determine the efficiency of Transfection in order to normalize data obtained through another experimental method. I utilize a pIRES2-EGFP vector with my gene inserted upstream of the IRES sequence. Within the cell a single transcript containing my genes sequence as well as the EGFP sequence is produced. The Experiment: Transfect HEK 293 cells with a pIRES2-EGFP vector that has my gene of interested inserted into the MCS (upstream of the IRES sequence). The cells are run through two experiments: 1. A maximum binding (Bmax) value is obtained through whole cell binding using a competition assay with eight concentrations of radio labeled ligand verses saturated cold ligand. 2. FC is used to determine EGFP fluorescence using a standard EGFP bead provided by BD bioscience as a standard. The Analysis: The idea was to use the values for each experiment in a ratio of Binding over EGFP. The Bmax value is determined through Scatchard plot analysis. The Question: I am using the EGFP value obtained for a single experiment but I am not sure how to remove the auto-fluorescence of untransfected cells. I am currently trying to use the population comparison function within FlowJo, which allows the use of a control, but I am not sure what value is the important piece of information. I have tried to attach a sample of a comparison. Any help would be appreciated. Samuel <<Picture.doc>> ------------------------------------------------------------------------------ Confidentiality Note: This message is intended for use only by the individual or entity to which it is addressed and may contain information that is privileged, confidential, and exempt from disclosure under applicable law. If the reader of this message is not the intended recipient or the employee or agent responsible for delivering the message to the intended recipient, you are hereby notified that any dissemination, distribution or copying of this communication is strictly prohibited. If you have received this communication in error, please contact the sender immediately and destroy the material in its entirety, whether electronic or hard copy. Thank you. ------------ Visit us online at our award-winning www.clevelandclinic.org for a complete listing of Cleveland Clinic services, staff and locations from one of the country's leading hospitals. ============================================================================== This attachment - 'Picture.doc' - 21.50 KBytes - can be viewed at http://www.cyto.purdue.edu/MD-parts/5b5b99ac1e692e4be8771663739a2d7b4b67b6fa.docReceived on Thu Sep 30 15:58:00 2004
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