Fixation/Ab-binding
Kevin G Waddick (waddi002@maroon.tc.umn.edu)
Mon, 7 Jul 1997 10:25:00 -0500 (CDT)
Hi everybody,
I am puzzled by a question that I hope someone can help me understand.
We are using a human lymphocyte cell-surface protein extracellular domain
that, through stable transfection, is being produced in insect cells.
Although this antigen itself is well-characterized, and it seems to be a
receptor protein, no counter-ligand has been definitely found yet.
Here is the problem. We have biotinylated the protein and are testing
a wide range of different cell types for the ability to specifically bind
it. When we test unfixed cells by flow cytometry, we either get no
staining (using either avidin-FITC or -PE) or when there seems to be some
staining, it is merely 1-10% of the primary or cell line cells with a
large range of intensities (starting at barely above the
control-determined +/- level). So far so good, however, when a guy here
did side-by-side fluorescence confocal microscopy, he used cells that
either were or were not formaldehye-fixed prior to treatment with protein
followed by the avidin-fluorochrome; the fixed cells of a particular type
showed a definitely positive fraction of ~50%, while the unfixed cells had
a positive percentage similar to the unfixed cells examined by FCM. When
previously fixed cells were tested using FCM, a tightly-focussed,
reasonably intense positive population of ~60% appeared, whereas in the
unfixed sample the positivity was only 5% with the usual scattered
intensity. The staining can be partially blocked using unbiotinylated
ligand.
I don't think the staining of the fixed cells is nonspecific because
cell types that were totally negative when unfixed remain negative when
fixed. It seems contrary to sense that if a cell has a specific receptor
for a ligand, it only becomes highly evident using fixed cells and is
questionable when using healthy, unfixed cells. Does anyone have
experience with this phenomenon? What does it mean?
Kevin G. Waddick, Ph.D.
Staff Scientist
Hughes Institute
2685 Patton Road
Roseville, Minnesota 55113
(612) 628-0598
(612) 628-9891 Fax