Kristi- We do not have experience with tetramers yet, but we have done some rare event detection on the XL. Here is my take on your questions: How do you setup and verify the instrument for accuracy in detection? You need an appropriate negative control. Be certain to acquire as many events for the negative control as you do for the experimental. If you are using multiple colors to gate on subsets, these need to be the same on your negative control and your experimental (only the tetramer is different). The results you get here will determine the nonspecific background and hence the sensitivity of the system. We have tuned some of our rare eventing such that no positive events are detected in our negative control when 3 million events are acquired. If you are only doing 2 or 3 color, acquire a channel as "unstained" (e.g. FL4, remember it still needs to be compensated). Autofluorescence is often "broad band". Events positive for the "unstained color" can be gated out in both the control and experimental tubes. This removes a potential source of noise. What is a reasonable total cell count to analyze for statistical accuracy? It depends on the frequency of your rare event and the amount of noise in your negative control. In a recent application where we were looking for labeled injected DCs in draining lymph nodes we acquired 1E6 events in triplicate. Triplicate determinations are especially useful because you can calculate confidence intervals about the percent positive. Be prepared to sit in front of the cytometer for a while. It also helps to collect "time" as a parameter. If the instruments burps during your long acquisitions you can gate out the bad data during off-line analysis. Is available disk space the only limitation to the size of a listmode file accumulated on the XL? Its not the disk space, but the amount of RAM. My XL came with 16MB. It was necessary to upgrade it to 64MB to do industrial strength rare eventing. I hope this helps, Albert D. Donnenberg, Ph.D. Interim Director Bone Marrow Transplant Program, UPCI Phone: (412) 624-9596 Fax: (412) 624-9624
This archive was generated by hypermail 2b29 : Wed Apr 03 2002 - 11:53:54 EST