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Dear Jeffrey,
we also use the ORPEGEN test.
1) The kit uses FITC labeled bacteria of homogegeneous size and pure with long
time stability.
2) The quenching dye has a higher affinity than trypan blue or crytal violet and
allows subsequent washings.
3) The lyse and fix step give a stability that the ready samples can be stored.
4) The PI counterstain allows to gate on leucocyte DNA in order to get rid of
platelet aggregates and bacterial or cellular aggregates.
5) The FITC label is pH dependent however this fact does not impair fluorescence
intensity within the time frame of 15 min as BODIPY labeled e.coli show the same
time course (BODIPY is not pH sensitive).
All together helps in a clinical setting (low day to day variation, robustness)
but you can in principle prepare everything by your own.
2) Cytoplasmic IgM
Dear ChienCheng,
we use for a long time the DAKO anti IgM (Fab2) FITC and An der Grub lysing
reagent. First wash whole blood 2x to get rid of serum, then use AdG protocol
and preincubate with rabbit serum before adding anti-IgM-FITC. Add mouse serum
and counterstain with CD19 or CD22. This procedure can also be adapted for
cytoplasmic light chain restriction in immunocytoma and plasmocytoma.
I hope protocols like this will be on the next Purdue CD. We will also discuss
this methods in our flow course (oct 12-17 in Mannheim).
regards
C. Thomas Nebe
Klinikum Mannheim, Faculty for Clinical Medicine, University of Heidelberg
68135 Mannheim, GERMANY, phone +49 621 383-3845, FAX -3819
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