From: Kroeger, Jodi (kroegerJL@Moffitt.usf.edu)
Date: Wed Feb 25 1998 - 11:44:29 EST
Dear Colleagues: I realize this is a topic previously discussed thousands of times, and I have attempted to search through the Internet archives to address this question for a researcher in my core lab. However, there are so many messages and so little time that I would be sitting for hours to find the specific reply to this question. So...I apologize in advance for the repetitive nature of this request for help. Any response will be greatly appreciated by me and many users of my facility attempting to do GFP and PI staining. Please reply to me and I will pass the info on to others. Objective: Analysis of GFP and PI in NIH 3T3 fibroblast cells. Current Protocol: Removal of adherent cells with 1X PBS/0.5M EDTA and scraping. Fixation with 1% paraformaldehyde for 10 minutes on ice. Cells stained in PBST at 50ug/ml PI and 100ug/ml RNAse A and kept over the weekend until analysis. Obstacle: Cells are clumping, which renders a clean cell cycle profile of GFP-positive cells impossible to acquire. Question(s): What can be done to prevent the clumping...is it related to the removal of the adherent cells? Are the concentrations of the reagents and times of incubation sufficient? Thanks Again, Jodi L. Kroeger for Tammy L. Bowman, Ph.D H. Lee Moffitt Cancer Center and Research Institute Tampa Florida
This archive was generated by hypermail 2.1.6 : Thu Jan 01 2004 - 17:35:13 EST
