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
