I am running a TUNEL assay (currently without PI) for a researcher that has preprepared it and brought it to me for analysis (I have some influence over the manufacturer chosen for the kit, but price tends to govern more than my input). The researcher is working with chondrocytes grown in a 3-D culture in agarose. The company has told me (if I understand them correctly - no cytometrists on board to answer questions) that a neg will have 2 peaks on a single histogram and a positive will have 3. Currently I am not using any gate to look at this data, just total 530nm fluor, primarily because on FSC and SSC; first, I cannot get all possible populations on my oscilloscope at one time and have had so few cells available I could spare none to look under a fluorescent scope and second, I can find no information telling me that I need to use or not use an FSC/SSC or other gate (I'm not sure the company people understood what I was asking when I asked about gating). So my first problem is "To gate or not to gate?". My second problem is that what I do get on FSC/SSC scatter is odd looking; 1 population with very,very high SSC off-screen, 1 population with very,very high FSC off-screen and a third population of either what appears to be a debris pattern increasing primarily in the FSC direction from the origin or a broad FSC spread with the same SSC throughout (from the origin, on a 2-D plot - a long thin population). I do realize that free apoptotic bodies are in this equation and may indeed be reason enough not to use an FSC/SSC gate, but I would like to hear what others would do in the same situation. My third problem is that some negatives have only 1 peak and some positives have only 2 and in many cases the second peak percentage on the positive (with or without a 3rd peak) increases above the second peak percentage on the negative, from what I gathered that the company was telling me, I do not bother with second peak percentage increase only the percent increase in the third peak and that causes me some concern, esp. when there is no third peak. I would greatly appreciate any and all input, questions, etc. I will fax any info that would help to explain my dilemma. You may phone or email me directly at the phone # or email address listed below. Thanks in advance, Jeanene Swiggett ------------------------------------- Name: Jeanene Swiggett Institution: Carolinas Medical Center; Charlotte, N.C. E-mail: jswigget@carolinas.org Phone: 704-355-7269 Date: 5/13/97 Time: 8:33:42 AM This message was sent by Chameleon -------------------------------------
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