Hi Carolyn, The death-related clumpy stickiness could be due to DNA from the dead cells - try adding DNAse to prevent the re-clumping after filtration. Ray > From: Carolyn Jefferiss <prscmj@bath.ac.uk> > Date: Tue, 19 Sep 2000 13:23:39 +0100 > To: Cytometry Mailing List <cytometry@flowcyt.cyto.purdue.edu> > Subject: clumping and sieving > > > Dear All, > > Has anyone tried using "Accumax" from TCS Cellworks at all in flow > cytometry? It is supposed to be an anticlumping reagent, recommended > for using for cell counting, which requires little or no Ca++ or > Mg++. The cells are not suitable for culturing after disaggregation > in this reagent. Anyone? > > My little bit on clumping on the sorting front; > We sort cultured marrow cells which can be horribly clumpy and sticky > (hence my wondering about Accumax) and we sort with a suspension at > 10x10e6cells/ml having filtered the cells immediately before putting > on the machine and the proportion of dead cells definitely affects > the amount of clumpiness which develops. We don't put on more than > 2ml at a time, and during a run take off the tube and give it a very > gentle mix if it is really settling out, rather than using the buzzer > on the machine. They can always get a second pass through a filter. > The on-line thing sounds like a big faff if you get it clogged up so > we've never done it. Since persuading everyone of the need to filter, > before running each and every sample, we haven't had ANY blockages. > (Famous last words). > > Carolyn Jefferiss > > Carolyn Jefferiss Ph. D. > Pharmacy and Pharmacology > University of Bath > Claverton Down > Bath BA2 7HY >
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