t 07:28 PM 12/16/94 -0500, you wrote: > >Dear Flow-ers, > >My colleague and I would like to set up a test for the expression of >cytoplasmic IgM for differential diagnosis of B-ALL and would >appreciate any comments /advices /references on the problems that we >encountered, mainly how would one tell the difference between a surface IgM >versus a cytoplasmic IgM? Can I somehow block the staining of surface IgM >or can I quench the surface staining as is being done with phagocytosis >tests? And is there any good controls for the tests? > >We are also evaluating the possibility of performing a cytoplamic CD3 >staining for diagnosis of T-ALL and, quiet possibly use it for monitoring >of minimal residual disease. We have the same concerns for this >test as we mentioned for cytoplasmic c-mu and would appreciate any comments >/advices /references. > > >Warm regards, >Austin Lin >Core Facility Laboratory-Flow Cytometer, >Cheng Gung Medical College >Tainan, Taiwan > Austin: We have used a two-color method to stain both surface and cytoplasmic IgM in mouse splenocytes. 1. Incubate with biotinylated-anti-IgM in PBS-0.5% BSA-0.01% Azide (PBA) 2. Wash 3. Fix in PBS with 1% ultra-pure formaldehyde (Polysciences). 4. Wash 5. Fix in 70% ethanol (OR perform all of the following in PBA with 0.5% saponin) 6. Incubate with anti-IgM FITC and strepavidin-PE in PBA 7. Wash 8. Fix again in PBS with 1% formaldehyde 9. Analyze The cells with surface IgM (and some cytoplasmic IgM) will be double-positive. The cells with stictly cytoplasmic IgM will be only FITC-positive. Jeff Clapper Cell and Hybridoma Facility Iowa State Univeristy 515-294-8504
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