Dear Volker, As far as I known there is no "golden" standard for preparing cell suspensions from fresh solid tumours for multi-colour purposes. You can find many different protocols in the literature. We share your experience with the debris. However, it depends on the kind of tumour you try to dissociate and the enzyme mixture you are using. We based our protocols on the work of John F. Ensley et al: Solid tumour preparation for flow cytometry using a standard murine model. Cytometry 1987; 8:479-487 & Solid tumour preparation for clinical application of flow cytometry. Cytometry 1987; 8:488-493. Not without success: Cytometry 2000; 39:96-107, Genes Chromosomes Cancer 2000; 28:173-183, Cytometry 2000; 41:73-80, J Exp Med 2000; 191:961-975. We use a DNA stain at high concentrations to "get around" the debris. This has several advantages: you can gate on single nuclei using your pulse processor (subtracting most of the clumps and debris) and activate this gate during acquisition in your dot plots. This will clear up your sky tremendously. It also provides you with DNA ploidy information simultaneously (very helpful in identifying tumour cell subpopulations). A disadvantage is that you have to sacrifice one FL channel for your DNA histogram and that there are more spectral cross-talk problems, but these can be solved. Good luck. Willem E. Corver, BSc, PhD Leiden University Medical Centre P.O. Box 9600, Building 1, L1-Q 2300 RC Leiden The Netherlands -----Original Message----- From: Eckstein, Volker [mailto:Volker.Eckstein@med.uni-heidelberg.de] Sent: maandag 2 september 2002 11:46 To: cyto-inbox Subject: single cell preparation Hi everybody, I know a lot of people are still on holidays but nervertheless I am posting my question again. I've got no replies yet. Dear experts, is there anybody who has experience or a good working protocol in preparing single cells from stomach carcinoma tissue or kidney carcinoma tissue for further flow cytometric anaylsis? I have a client who started to do this but the first attempts looked - let's say not as what we assume and expect is a nice single cell suspension for flow analysis. A lot of debris and a lot of aggregates also seen during counting the cells on the microscope. Even after filtering the 'suspension' I can't identify a certain cell population. And there also is no difference between permeabilized (for cytoplasmic antigen staining) and nonpermeabilized 'cells'. I appreciate any help Many thanks in advance Regards Volker (a little bit stressed about this isssue) Volker Eckstein PhD Dept. of Internal Medicine V Medical school of the university University of Heidelberg GERMANY volker_eckstein@med.uni-heidelberg.de
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