From: ctnebe (ctnebe@t-online.de)
Date: Thu Jul 11 1996 - 12:03:18 EST
1) Phagocytosis 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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