Safe sorting of GFP-transduced live cells for subsequent culture using a modified FACS vantage

Background: A stream-in-air cell sorter enables rapid sorting to a high pur ity, but it is not well suited for sorting of infectious material due to th e risk of airborne spread to the surroundings. Methods: A FACS Vantage cell sorter was modified for safe use with potentia lly HIV infected cells. Safety tests with bacteriophages were performed to evaluate the potential spread of biologically active material during cell s orting. Cells transduced with a retroviral vector carrying the gene for GFP were sorted on the basis of their GFP fluorescence, and GFP expression was followed during subsequent culture. Results: The bacteriophage sorting showed that re biologically active mater ial was confined to the sorting chamber. A failure mode simulating a nozzle blockage resulted in detectable droplets inside the sorting chamber, but n o droplets could be detected when an additional air suction from the sortin g chamber had been put on. The GFP transduced cells were sorted to 99% puri ty. Cells not expressing GFP at the time of sorting did not turn on the gen e during subsequent culture. Unsorted cells and cells sorted to be positive for GFP showed a decrease in the fraction of GFP positive cells during cul ture. Conclusions: Sorting of live infected cells can be performed safely and wit h no deleterious effects on vector expression using the modified FAGS Vanta ge instrument. Cytometry 37. 284-290, 1999. (C) 1999 Wiley-Liss, Inc.