Hi Steve, I have worked for a long time with whole blood and I never noticed autofluorescence interference unless in a few situations. Samples already labeled for over 2-3 days, cultured monocytes, eosinophils are always autoflurescent, CD8+ cells from some HIV+ patients and problems with the quality of my Fetal Bovine serum in experiments that use it as a macromolecular media. As far as erithocytes I only found autofluorescence when I fixed them before the procedure, using either PFA or GA (glutaraldehyde). If you can give more details about your protocol I will be happy to help you. I have worked with red blood cells with different protocols with no problem. Moreover, I have tested human as well as dog RBC. I am looking foward to hear from you, best regards, Olindo ---------- De: Steve Woodard[SMTP:steve.woodard@ibb.gatech.edu] Enviada: Terca-feira, 8 de Fevereiro de 2000 18:06 Para: Cytometry Mailing List Assunto: controlling autofluorescence red cells/whole blood Does anyone have experience in performing analysis of red cells in whole blood. I am encountering problems with the amount of autofluorescence associated with whole blood samples. When I look at FL1 VS FL2 plots of "Cells Only", the majority of cells are in the lower left quadrant. There are some cells that are found at a 45 degree angle to the lower left quadrant. It reminds me of a "comet". The problem is that the autofluorescence is interfering with stained cells that are double positives. In order to complicate matters, we are looking for 50 events out of 50,000 in the whole blood ( 0.1%). How is this type of problem usually solved ? I tried using trypan blue but there was only a minor reduction in autofluorescence. I came across crystal violet as a quencher but fixation of the whole blood is not an option. thanks in advance for your responses, Steve
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