Dear Flowers,Thanks for all the replies concerning CellCyt and single cell sorting followed by PCR. 1. Spin plates after deposition, use a max volume in wells, PCR is not always 100% 2. Use a 3 drop envelope (or 4) and a slow sort rate 100-300 events 3.If you are going to grow cells after sorting gate out dead cells with PI,success rate 50-60% 4.Use large beads for set up similar in size to cells to be sorted. Stream /drop stability can be affected by larger cells. Then check sorting with cells. 5.Counter mode may need a longer drop delay than Norm R, this can be an issue with drop delay less than 13.5. 6. One cell per well is not always consistent but several replies stated they would expect to get 90-96% of wells with one cell. I run my Vantage at 13psi.sheath pressure,with a 70µ nozzle, DDF=29000 drop delay=11-13.I set up with fluorescent beads then do test sorts using fixed cells (at intervals of 0.2) Most sorts are 93-97%,best to date 3% cell line back at 98%. Today I tried Clone cyt again using 10µ beads,and a 3 drop envelope,sort rate 200per sec .Sorting onto slides, most wells had only one bead ie 11 out of 12wells, 14/14,12/14 so I am convinced the machine works the problem probably lies with cells sticking to the sides of the wells or the PCR (as some of you pointed out) Thanks for all help Geoff Morgan
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