|From:||Gayle Callis <firstname.lastname@example.org>|
Two sources of how to do cell smears: Pharmingen (Cytokine Cytochemistry)
and R&D Systems.
This is the R&D Systems
Harvest cells, wash twice with cold wash buffer (400g/5 min) to remove
Cell concentration should be 1-5 x 10 to the sixth cells/ml
Put 10 - 15 ul cell suspension on plus charge slide, allow cells to adhere
electrostatically in a monolayer for 10 min (RT, humidified chamber) do not
air dry, then fix with 50 uo ice cold NBF or paraformaldehyde, to fix
cells, fix in humidified chamber for 20 min (time could be optional), wash
3 X to remove fixative.
This works fine for PFA or NBF fixation since cells are never air dried,
but not well for acetone fix on our cell markers.
We prefer to make cell block, washed 3 times in culture media or buffer, at
approx 2 x 10 to the seventh cells, spun down in a 50 ml centrifuge tube,
remove all supernate, introduce OCT carefully on top of cell pellet, snap
freeze, then pop block out, double embed in OCT, do frozen sections (2 - 4
um). Cell morphology is superior and more concentrated than a smear. Air
dry frozen 30 min to overnight, fix in fixative of choice, run with IHC.
Whatever you do, don't use 15 ml tubes, too narrow and can't get block out!!
Smearing cell suspensions followed by air drying was a disaster, touch
preps were just as bad and dislike tedious preps etc recommended by R&D
(ala Anderssons/Litton publications) and BD Pharmigen.
You can do cytospins, but very gently, not at high speed or for long times
since cytoplasm rounds into a tight rim around nucleus, not morphologically
Pharmingen recommends incubating the fixed cells 10 min in 1% BSA, air
drying and freezing down for future IHC. I presume carrier protein here
cryoprotects the cells a bit by coating them. They have a booklet on these
protocols, as does R&D (called Cytokine detection by IHC) on website.
Veterinary Molecular Biology
Montana State University
Bozeman MT 59717-3610
<< Previous Message | Next Message >>