Hi Beth, we have worked with BAL and performed flow cytometry - both in normal subjects and those with COPD. The COPD patients occasionally have slightly gloopy BAL, but on the whole, I have found it to be not too bad. We do not filter or use DTT, so as to interfere with the sample as little as possible. Some people will filter like you have done, and you may have to do that on occasion, tho' we have avoided it. We did have one BAL which was very gloopy, and even after centrifuging, we didn't get good separation. However, what we did was simply to lift out the gloopy, mucusy bit. Yes, we probably lost some cells, but we could not think of any other option. I would suggest caution with DTT, as it may interfere with some cell surface markers, and there is some literature on this. A recent study in the ERJ (below) though done on blood, would hold this up, and there are other papers: D. Loppow, M. Böttcher, G. Gercken, H. Magnussen, R.A. Jörres Flow cytometric analysis of the effect of dithiothreitol on leucocyte surface markers Eur Respir J 2000; 16: 324-329 I have seen the reply by Bruce Greig in Nashville, and that would seem eminently sensible. Best of luck with your work. Martin ---------------------- Martin Kelly Department of Clinical Biochemistry, Institute of Clinical Science, Grosvenor Road, BELFAST. BT12 6BJ Tel No: (028) 90263267 On Fri, 22 Sep 2000 16:51:26 +1000 Rees Beth <beth.rees@dchs.tas.gov.au> wrote: > > Hi Flowers > > Here's one for the clinical flow cytometrists - how do you separate the > cells from a slimy snotty goopy bronchial lavage ( have I totally put off > the non-clinical cytometrists yet?). I have tried diluting it and flushing > it through a 100um filter, but as soon as I centrifuge it the gunk just > condenses again. > The microbiologists use something called mucolyse (dithiothreitol) which > reduces mucoprotein disulphide bonds, but I'm a bit scared to use this in > case it interferes with the cell surface in any way. > > Any suggestions much appreciated. > > beth.
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