293 cells are among the easiest to clone that I've ever done--they are so hardy. Although I haven't done this in 8+ years, from what I recall we usually sorted them into 20% serum (growing them in 10% usually), but I don't think that matters so much. On a typical 96 well plate with single-cell sorts, we would get anywhere from 50-90% of the wells coming up with colonies. We always tried to keep the cells as happy as possible by pre-incubating the sort tray in the incubator to equilibrate temperature and gas (and pH). Of course, we usually stained the cells on ice, so they were already chilled; had we kept them at room temperature for the duration, I bet our recovery would have increased some. We did not find the presence of azide during the staining time to affect the cells (as long as they are kept at room temp or below); the dilution once single-cell sorted into the tray is so huge that residual azide is irrelevant. However, it can't hurt to leave this out as well. mr
This archive was generated by hypermail 2b29 : Sat Mar 10 2001 - 19:31:25 EST