Summary Document
This document is a summary of message from the Purdue University Cytometry Laboratories Cytometry Discussion list.
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Original Question:
Does anyone know a good method for disrupting doublets for sorting without
destroying the cells themselves?
We have had this problem with macrophages, monocytes and in some cases DCs.
The doublets result in a major reduction in recovery, any ideas would be
welcomed.
Kylie Price
Staff Scientist Flow Cytometry
The Malaghan Institute of Medical Research
The Central Services Building
Victoria University Campus
Entrance 7
Kelburn Parade
Wellington
New Zealand
Ph: 64-4-4996914, ext 850
Fax: 64-4-4996915
E-mail: kprice@malaghan.org.nz
Web: http://www.malaghan.org.nz
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Replies:
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* I am currently working with human cell lines DU-145 and HT-29, both
of which aggregate in solution. We syringe the suspension two times
through an 18 gauge needle before counting; it seems to take care of
most of the doublets without harming the cells. Hope this helps!
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* You could try sorting your cells straight from Accutase which I have
done with good preservation of viability and downstream function.
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* There are several tips for preventing clumping. Using EDTA at up to 5mM
concentrations may help prevent cation dependent cell-cell adhesion. Do not
keep the cells at too high a concentration either (no more than 5-6
million/ml). Likewise using Ca/Mg++ free buffers will also help. Remember
adding serum to your buffers will replace the Ca/Mg++ so if you need a
protein support try using BSA instead. If your cells are clumping due to
dying cells, the addition of DNAse II (10u/ml) may help. Of course you could
filter your sample through a nylon mesh but the important thing is to
prevent clumping so you do not lose too many cells.
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* I use 2-5% BSA to coat the cells. I think it also helps protect the cells,
when sorting, from the degassing effects at the
nozzle exit. If they are really clumpy I add some DNAse [assuming the cells
are alive].
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* Have you tried edta or a low conc. of trypsin?
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