Fred Menendez wrote: >A researcher at our institution wants to use flow cytometry to measure the >infection rate of RBCs with malarial parasites by measuring the malarial >DNA content of the RBCs. He would use PI and possibly AO as the >fluorochromes. He also would like to differentiate at what phase of the >cell cycle the majority of the cells are in (?) by determining the number >of cells in G0/G1 vs G2/M. > >While in theory this sounds feasible, and while I have done just such >measurements on tissue and WBCs, I have no experience with the measurement >of parasitic or RBC nucleic acids. Of course, with the exception of >reticulocytes (which can be measured by flow using AO) the vast majority of >circulating RBCs contain no nucleic acid. As such, I have a number of >technical question I need to answer before we begin these experiments. AO is a poor dye for measuring either malaria parasites or reticulocytes; background fluorescence is high and discrimination between DNA and RNA is poor. PI would not be wonderful either; the RBC's would have to be fixed and RNAse treated. Malaria parasites stain well with Hoechst dyes; if you have a UV source available, that would probably be the best method to use. Thiazole orange is also a good dye for malaria parasites, and, of course, excites well at 488 nm, but is not DNA specific. NRBC's have a great deal more DNA than malaria parasites; there shouldn't be a problem discriminating nrbc's from parasitized cells, but the signals from parasitized cells may be small enough so that you get very broad peaks due to poor photoelectron statistics. I suspect the cell cycle distribution of malaria parasites is not going to look much like that of eukaryotic cells. I haven't looked for recent literature on flow cytometry in malaria although I will be doing so in the course of putting together the 4th Edition of Practical Flow Cytometry. It shouldn't be a hard search to do. I can get you the older references, but you should probably look for newer ones. -Howard
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