Dear Sarah, I would recommend you to avoid any sample manipulation including erythrocyte lysis at all and use a vital nuclear dye instead (LDS-751 from www.exciton.com or www.probes.com) with triggering on its fluorescence. For me it works well. For reference see: Blood Cells 1991;17(3):585-602; discussion 603-5 and J Immunol Methods 1997 May 26;204(2):175-88. -- Karel Drbal Laboratory of Leukocyte Antigens Institute of Molecular Genetics Academy of Sciences of the Czech Republic Videnska 1083 142 20 PRAHA 4 Czech Republic, Europe > Sarah Lawson wrote: > > Dear All, > > HELP!!! > > This is probably a very basic problem, but I hope someone out there > can offer me some advice. > > I am studying minimal residual disease in acute leukaemias using > triple colour flow cytometry and a stain and lyse technique. > > My problem is with lysing my specimens adequately, in that several > specimens have not lysed well recently resulting in them being > unanalysable on the flow. This has happened once or twice previously > and I put it down to something I was doing wrong in my early days in > flow cytometry! > > However, over the past 2 days I have had problems with ALL of my > specimens. I have not changed my lysing reagent or technique in any > way, and the problem occurs with both commercial and home-made lysing > reagent. It is also occurring with both peripheral blood and bone > marrow specimens. > > I have several questions: > > 1. Has anyone had similar experience, and if so how have they overcome > it? > > 2. Any tips or pointers to getting these stubborn cells to lyse? > > 3. I often have specimens with low cell counts, and so usually add a > larger volume of the specimen to the antibodies. This obviously means > that there are more red cells present to lyse. Is it okay to just add > a greater volume of lysing reagent? If not, how does one overcome the > problem of low counts? > > I would be most grateful for any advice anyone can offer. Speedy > advice would also be appreciated as I have more specimens tomorrow!!. > > Thanks in anticipation. > > Sarah Lawson > (isaac.lawson@btinternet.com) > >
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