Dear all, I would like again to thank everybody who mailed me their B cell isolation methods. I've had several requests for these methods so please them below: 1) An inexpensive method to isolate human B-cells from peripheral blood is to: 1) isolate lymphocytes using hypaque-ficol, 2) deplete T-cells using neuraminidase treated Sheep RBC (same technique used to make T-cell rosettes), and 3) deplete monocytes by incubation at 37 deg. C in plastic flasks (monocytes stick). Although I haven't done this in about 20 yrs, the cells were, as I remember >95% sIg pos. Hope this helps? 2) You might have better luck using magnetic beads. Couple the C3d to the beads, expose to your cell mix, then use a magnet to hold back the beads, wash them, and then separatre the cells from the beads, e.g. by competing with soluble C3d. Alternatively, you can use negative selection - add mouse monoclonal antibodies to everything except the cells you want, use anti-mouse IgG couples magnetic beads to remove the labeled cells, and you'll have the ones you want without having to elute them. We had good luck with this technique with separating memory from naive cells 3) You could try putting your C3d on washed erythrocytes, then positive-select by rosetting and centrifuging over Isopaque-Ficoll. This is preferable to an affinity column such as you're describing - likely to give better purities. Even so, you won't do as well as you would by MACS. Have you thought of Negative selecting - removing the granulocytes, T cells, NK cells, etc?. Rosetting is very good for that. General considerations and some specific recipes are in my chapter in Weir's Handbook of Exptl immunology, 4th ed (chap 55). Written in 1986, so doesn't emphasise magnetic metohods as much as I would now, but otherwise it has lasted reasonably well. Are you sure human C3d serves as good ligand to rabbit, marmoset, and rhesus monkey CD21? I don't know at all, but cross-species reactions don't always oblige as well as one would like. 4) How about the Dynal magnetic beads (http://www.dynal.net/). The beads are much larger than the MACS ones and the magnet you need doesn't cost a fortune. (However the beads cost about the same as the MACS beads). Biggest disadvantage compared to MACS you cannot run cells labelled with Dynal beads through a cytometer! (but there are ways to get rid of them for cytometry). 5) You could try panning, an old tried, but true methodology. 6) The "old-fashioned" way of collecting human B cells is to pass a lymphocyte prep (i.e. after Ficoll separation) over a nylon wool column, but I don't know if this works for other species as well. The B cells adhere and the T cells pass through. The B cells can be collected by forcibly passing medium through the column. Specific details should be readily available in B cell references or in methodology textbooks. Let me know if you can't find a protocol; I should have one kicking around here somewhere Thanks again, Louise Dr. Louise Affleck Senior Scientist Protein Function Group AdProTech Ltd Unit 3, 2 Orchard Road Royston Herts SG8 5HD Tel: +44 (01763) 226090 Fax: +44 (01763) 226021 http://www.adprotech.co.uk
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