To answer a few questions from David L. Haviland: I analyze calcium flux in lymphocytes on a regular basis on a cytometer, and while tough, it is doable. I do not think that any of the physical differences you mention are relevant; in fact, I have compared dose response curves of a tumor infiltrating lymphocyte (TIL) line to a chemokine, and they were identical. First of all, fluorimeters use ratio measurements, either fura (dual excitation, single emission) or indo-1 (dual emission, single excitation). I cannot overstate the importance of ratio measurements. The spread of loading within lymphocytes is quite large, but on a bound vs unbound probe scattergram, the events are a very tight line. If one were to look at either parameter of the ratio alone, there is no way you could detect a small number of responding cells, or even a small response of a large number of cells. The calcium rise after ionomycin is huge, and much greater than any physiologic ligands I have tested (cross-link BCR, TCR, or chemokines). It is a good place to start to let you know that cells are loaded, but not worth anymore than that. With Jurkats, it is better to look at cross-linking the TCR. Cells do move in a fluorimeter, there is a stirrer; a fluorimeter is looking at many events at once, and there may be some amplification there. At least I think so, but those with greater knowledge of the physics may know better. It is possible that a small number of events can be seen in a fluorimeter, if the signal from those events is high enough. One example is that many studies show "PBMC" responding to a particular chemokine. In fact, it is only the monocytes, which make up about 25% of the total. Fluorimeters are cheaper and easier to use than a cytometer by far. If one has a homogeneous population of cells (e.g. transfected 293) , a fluorimeter is the best choice, because there is no reason one would be doing subset analysis. Finally, molecular probes has a probe, I think it is "Fura red" that increases in fluorescence when not bound to Ca. It emits at 670nm. You can load your cells with fluo and fura red, and use a program such as multitime to ratio the two signals if you want. Ronald L. Rabin, M.D. Laboratory of Clinical Investigation National Institute of Allergy and Infectious Diseases National Institutes of Health Building 10/Rm 11N228 MSC 1888 10 Center Drive Bethesda, MD 20892-1888 Phone (301) 402-4910 FAX (301) 496-7383 email: rr84g@nih.gov
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