Greetings: To make a long story short, I've managed to get Ca2+ flux assays working using Jurkat cells and Fluo-3AM. It took some doing and fiddling but with either Ionomycin or A23187, we can see a full log peak rightward peak shift in Fluo3AM loaded Jurkat cells. I would like to hope that we open that range up with further optimization. With the next set of experiments, came a plethora of questions. I attempted to load every tissue culture cell line currently on my shelf using the same parameters as I did for the Jurkat cells. Using the same inonophores (A23187 and Ionomycin) as above, I had hoped to see differences between control and maximal values. As expected from the Indo-1 experts that I've contacted, the response of the cells with Fluo3AM was varied to say the least. Unlike the Jurkats, the differences in the cells (COS1, HL60, K562, Raw264.7, and U937) between control and ionophore treated cells hardly differed by more than half a peak shift. In the case of Raw264.7, there was *no* peak shift. This differing response could be due to the unloading of Fluo3AM by the MDR Transporter. The question that came up on here a week or so ago is why do researchers see a response when using fluourimeter rather than a flow cytometer. We brought this up in our lab meeting and had an odd thought. In the flourimeter, the cells aren't moving as with a cytometer. So would not the fluorimeter, see and record any change from any number of responding cells? Going further, would a fluorimeter record a Ca2+ change in say 5% of 20,000 cells? (Never having used one yet... I can't say for sure.). In contrast, the much larger population of cells is moving through the cytometer and thus to see a change, would not a larger number of loaded cells must likewise respond to see a significant peak shift? I'm not convinced it is a matter of sensitivity between the two machines. Could it be more of a question that one doesn't need *as many* cells to respond in a fluorimeter as oppossed to a cytometer? It just seems odd that Ca2+ measurements in a flow cytometer mainly employ lymphoid cells. Yet, folks that do Ca2+ studies on various transfected cells seem to use a fluoimeter. I'm just trying to understand why wouldn't I see the same result with both machines using the same cell type and mediator? Thoughts? David ============================= David L. Haviland, Ph.D. Asst. Prof. Immunology University of Texas - Houston, H.S.C. Institute of Molecular Medicine 2121 W. Holcombe Blvd. Houston, TX 77030 Internet:"dhavilan@imm2.imm.uth.tmc.edu" http://www.uth.tmc.edu/`dhavilan Voice: 713.500.2413 FAX: 713.500.2424 "A conclusion is simply the place where you got tired of thinking." =============================
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