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1. We often precoat our polystyrene tubes with 10% (inexpensive) serum in
PBS to avoid having lymphocytes stick to the bare plastic (0.5 ml, 15 min
or so, then aspirate out). (It is documented in the literature that cells
adhere to bare plastic or glass by a rapid, temperature independent, and
non-detachable mechanism, probably direct plastic protein interactions.) If
the tube had serum-containing medium in it for a few minutes before the
cells are added, then this should not be a problem. But if the cell
aliquot is added to the tube first, we precoat.
2. Of course macrophages love that fibronectin coating produced by serum.
As an alternative, you might try precoating with 0.5% BSA in PBS.
3. Keeping cells on ice would undoubtedly help a lot.
4. At room temperature, we're able to prevent B LCL aggregation, even when
activated with PMA, by putting a tube containing 0.5 ml cells at about
a 15 degree angle from the horizontal in a test tube rack on a roller which
rotates the tube roughly once per second (around the long axis of the tube,
not end over end). The same might work for macrophages.
Good luck!
/*- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -
Eric Martz, Professor of Immunology emartz@microbio.umass.edu
Dept Microbiology Voice: 413-545-2325 FAX: 413-545-1578
Morrill IVN 203, Box 35720, Univ Massachusetts, Amherst MA 01003-5720
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