Netrophil isolation problem

Bryan Heit bjheit at ucalgary.ca
Wed Nov 15 11:55:31 EST 2000


I'm having a problem isolating neutrophils.  I'm purifying neutrophils
from human blood using dextran sedimentation, followed by hypotonic lysis,
with final isolation using fycol-hypaque centrifugation.  My problem is
with the dextran sedimentation stage.  

I'm using 6% dextran (15g dextran + 2.25g NaCl + 250ml ddH2O).  What
should be happening is a sedimentation lasting approximatly 30min.  This
should result in a layer of RBC free supernatant with leukocytes suspended
in it.  what's happening is sedimentation takes over and hour and I get a
supernatant with almost no cells in it at all.  I've attempted to draw off
the supernatant as it forms, but I still end up with no leukocytes in the
supernatnat.  Does anyone know what's wrong?

Bryan







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