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

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