CD4+ purity - (Dec/24/2005 )
I have been using the CD4+ T cell isolation kit II (Miltenyi) to isolate untouched CD4+ T cells. It works by negative selection (i.e. depletion of non-CD4+ cells). I usually begin from 10x10^6 cells and pass the cells through MS column.
However, the yield is usually 30-35% (of initial number of MNC) and purity ranges 80-85%. Is this the usuall yield of negative selection systems? CD4+ T cells are ~50% of MNC!! How should I increase CD4+ purity?
Especially the purities you get seem to be a bit low. With the CD4+ T cell isolation kit II we at Miltenyi Biotec normally reach purities above 90% and recoveries over the column step above 85%, provided that the starting material is of a good quality and from a healthy donor. Recovery in this case refers to the recovery of CD4+ T cells (number of CD4+ T cells in positive fraction/ number of CD4+ T cells before column step * 100). To get a good idea about the actual recovery, it is useful to determine the number of CD4+ T cells in the starting material, as even with healthy donors it can vary considerably (20-40%).
A lot of different factors can lead to low purities e.g. shortened labelling time, too low temperature (on ice instead of in the fridge) or increased total labelling volumes. To find the possible reason in your case it would be very helpful to get more information about the protocol you use. The easiest and fastest way would be to contact the Miltenyi Biotec technical support team in your country (www.miltenyibiotec.com ).
I hope you find these suggestions useful.
Miltenyi Biotec GmbH