Isolation of Kupffer cells, any suggestions?

Buxton Buxton at manga.demon.co.uk
Tue Apr 21 12:59:21 EST 1998


Hello,

I'm a phD student at the Free University of Amsterdam and I'm working on the
isolation of Kupffer cells (liver macrophages) from Wag/Rij rats. This
isolation is however a bit of a problem:

I have a problem with the purity of the isolated cells: only 85%. (using the
elutriation technique). Besides, I only get about 5 million Kupffer cells,
which not enough for substantial experiments.

Currently I'm isolating them by cutting the non-perfused liver into little
pieces, incubation the tissue pieces for 40 minutes at 37 degrees with
pronase and DNase. This cellsuspension is subsequently centrifuged on a
Nycodenz gradient to get rid of parenchym and erythrocytes. In the last step
the interfase is elutriated and the Kupffers cells should be in one of the
fractions having a purifty of 95%.
I've altered the amount and types on enzymes used, the elutriation
temperature, the buffer used for elutriation, and many other things, but I'm
still having a problem in the elutriation step.

If anyone has experience using the cetrifugal counterflow elutriation
technique (and especially concerning Kupffer cell isolation), could you give
me some suggestions? E-mail as well as public please!

Thanks for your time.
Anneke





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