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 Dear Fred, We have performed several studies on routine malaria evaluation and currently use thiazole orange mainly for malaria culture quantitations. The TO is made up at 1mg/ml of methanol as a stock solution and then used in working solution comprising 0.05uL of the stock in 1mLs of PBS. Use ~1 mL working solution per test. We buy the TO from Aldrich, cat number 39, 006-2 and it comes in 250mg or 1g sizes and costs equivalent of ~$20.00 for the smaller size . In our experience, nucleated RBC and retics are only problematic for clinical studies, but even then the parasites are seen as a discrete peak within the background of retics, and the DNA of the nucleated RBC is easily discriminated. Obviously if there is a marked retic response due to haemolysis then seeing the parasites is less easy. A good reference is Makler MT et al, from Cytometry 8: 568-570, 1987. Good luck Debbie Dr. Debbie Glencross MD MMed South African Institute of Medical Research and Wits School of Pathology Tel: (011) 489 8540 Fax: (011) 489 8589 Alternate email: glencross@mighty.co.za
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