MACS cell purification
amcshea at world.std.com
Thu Apr 7 08:35:35 EST 1994
Gene Holowachuk (geneh at CSCNS.COM) wrote:
: Hi netters,
: Our lab is interested in using the MACS/MiniMacs from Miltenyi Biotec for
: the rapid isolation of a rare subset of rat lymph node cells representing
: <1/10,000 using specific mAbs we have developed. Any
: recommendations/comments from those with experience with this system would
: be appreciated.
I have used this system a fair amount with success. First things-
I hope you dont requre loads of viable cells at the end, because the
viablity tends to suffer up to the tune of 50%, may be cell type
dependent I suppose, but nevertheless can be used and done easily under
sterile condtions. Why use this system for 1
cell in 10000- this is fairly needle in haystack'ish, a FACsorter might
be better if you can find one with enough throughput.
To get this sort of purification your going to need to have effective
washing, I would try using the smallest column (A2?) and a yellow needle
for the final wash. I can email you a more detailed protocl, but its at
work and I'm not at the mo. What you have to balance is the shar and the
field strength of the magnet where the cell is suspended, this is tricky
and depends how much non-spcific binding you get with unwanted cell
types. I would recommend find a FACscan, stain your cells with primary
antibody and then follow up with a biotylated second, if the primary
isnt, and use SA FITC, then microbeads. Then load the column and elute
using gradually increasing needle guage size and assay each fraction on
the FACs ( all though if its one in 10,000 yuo going to have o look at
like 200000 events?!). If you find your cells use the smallest guage
possible to wash, the more shear, the more unhealthy I presume it may be
for the cells.
Feel free to email me if I can be of any help!
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