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In response to Denis Snider's question for a procedure to isolate RNA from
sorted G2 phase cells.
You best fix your cells by slowly pipeting a suspension of cells into 4
volumes of icecold methanol (to make a 80% final concentration) on a vortex
at maximal speed. Fixatives such as warm 3.7% formaldehyde, cold or warm
acetone, and methanol/acetic acid give poor cell cycle phase resolution.
The maximal speed of the vortex is critical; otherwise your cells will form
a lot of clumps, which may be mistaken for G2 phase cells. Since you want
to recover RNA it may be advisable to add an RNase inhibitor such as vanadyl
ribonucleoside (New England Biolabs) to your fixative.
We got best cell cycle phase resolution when we centrifuged the fixed cells
and resuspended the pellet in PBS (supplemented with RNase inhibitor).
After at least 15 minutes at room temperature, cells are centrifuged again
and the pellet is resuspended in staining buffer. The latter consists of
Tris/HCl pH 7.4; 154 mM NaCl; 1 mM CaCl2; 0.5 mM MgCl2; 0.1% Nonidet P-40;
RNase inhibitor; 200 to 500 nM SYTOX GREEN (Molecular Probes, Inc.; Eugene
OR; Tel: (541) 465-8300; FAX: (541) 344-6504). After at least 15 minutes of
staining your cells are ready for sorting. SYTOX GREEN is best excited at
488 nm and fluoresces maximally at about 523 nm ("fluorescein" channel).
Since cell clumps have a larger pulse width relative to their pulse height,
you can distinguish cell clumps from "true" G2 phase cells. You need to
pull forwardscatter through a pulse processor to perform this distinction
procedure.
The RNA isolation procedure has been described by Esser et al. (1995)
CYTOMETRY 21, 382-386.
I would not recommend to synchronize cells in the G2 phase since that will
disturb the pattern of gene expression. Zbigniew Darzynkiewicz is an expert
on this point.
good luck !
Martin Poot
martin@probes.mhs.compuserve.com
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