Re: negative control for cytoplasmic C-mu or CD3
Jeffrey A. Clapper (clap@iastate.edu)
Fri, 31 Jan 1997 09:08:45 -0600
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