Dear Everyone, Does anyone put DNase 1 in their sort sample, to help reduce clumping, to good effect? ALSO:- I enclose the answers to my previous questions for anyone else who is interested,as attachments, as some went out to the List and some came directly to me; and a brief summary below. And, I apologise if my questions seemed ambiguous. It proved hazardous to discuss "sorting" by two different methods! In retrospect it would have been clearer to refer to "isolating" on the minimacs and "sorting" by flow cytometry. I expect everyone would like a preliminary step to reduce the negative population so that Flow Cytometric sorting can be performed more quickly, by getting rid of the bulk of irrelevant cells, and this is one of the things I am trying to do. Asking these questions is me trying to save on finding the answers empyrically because beads are so horrendously expensive. 1) I intended to find out if the magnetic nature of the initial separation by minimacs would leave magnetic particles on the cells which would cause them to be attracted to the difflection plates on the flow cytometer. Others have done this and find no interference from the beads, although 'bead manufacturers' have intimated that if the cytometer has any magnetic valves ( apparently some do) beads may accumulate here. 2)Possible "loss" of label; I haven't been re-labelling the POSITIVELY selected cells with primary antibody; just adding conjugated secondary for flow cytometric analysis of purity. e.g. anti-glycophorinA coated beads for minimacsing, then anti-IgGFITC conjugate for FACS checking. The general expectation of purity obtained with magnetic beads from a magnetic column seems to be lower than sorting - that is, happy with 70-80% whereas I was hoping for something nearer the 95+% I've had with Flow cytometry. What puzzled me was how the unlabelled cells were present, given that they weren't magnetically bound to the column. Dead cells binding non-specifically to beads was one answer. Amount of antigen present on the cells was another. However, as others regularly obtained 90-99% positivity it is probably my technique which is reducing purity. (I follow the manufacturer's recommended wash procedure, but still don't know how much more I can wash the column.) 3) Sorting time: A useful ref. given; " Cytometry 12:268-274 (1991)" Also advice; for rare cells - use fluorescence trigger 'enrich' mode and a high sort rate; followed by a resort with scatter trigger, in 'Normal' mode and a more dilute sample.
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