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Ethidium-staining nuclei

Kevin A. Morano kmorano at umich.edu
Sat Jul 12 16:07:39 EST 1997


Hi Ted,

   Here's a protocol I've used for flow cytometry. It gives excellent
propidium iodide staining, and I suppose would be equally good for EtBr. 
PI is cheap, though, you may want to just go buy some.  It gives an
intense red stain under the fluorescent scope, highly concentrated in the
nucleus.

Kevin Morano


FACS Analysis of DNA Content in Yeast



1. Grow cells under desired conditions and harvest 1 OD unit (107 cells).

2. Resuspend cells in 70% ethanol and incubate 30 min at room temp with
rotation.
   Cells will clump together during ethanol fixation but will disperse
after resuspension in buffer in step 3. Cells may be stored overnight in
ethanol at 4šC.  Mix well before proceeding.

3. Wash cells once in 50 mM  sodium citrate and resuspend in 0.5 ml same buffer.
   Cell washing must be done gently with fixed cells.  Spin for 1-2 min at
2,000 g.

4. Add 20 ul 10 mg/ml RNase A (40 ug/ml final) and incubate 2 hours at 37šC.

5. Wash cells once with 50 mM citrate buffer and resuspend in 500 ul PI buffer.

6. Add 10 ul 5 mg/ml propidium iodide (PI) stock (after pulse spin) and
stand for 30 min on benchtop (100 ug/ml final).
   Maintain PI stock and samples in low light conditions.

7. Spin out cells and resuspend in 1 ml 50 mM citrate buffer.

8. Add 1 ul PI stock solution (10 ug/ml final), mix and keep on ice until
ready to analyze.
   Check efficiency of staining by microscopy using fluorescence and the
"red" channel.  Brightly stained nuclei should be clear, with little to no
background fluorescence.  Vortex mix or briefly sonicate immediately
before FACS to separate cell clumps.

9.  Prepare 1:10 dilutions of stained cells and put 300 ul into 0.5 ml
pop-top tubes for use in the flow cytometer.
   Cell number  will dictate flow rate, and the slower the better. 
105-106 cells/ml work best,

PI buffer: 50 mm sodium citrate,  pH 7.0, 10 mM NaCl, 0.1% Nonidet P-40
PI Stock:  5 mg/ml in 50 mM sodium citrate, pH 7.0, filter stock before
use and keep      frozen.

Reference
 Kikuchi Y; Oka Y; Kobayashi M; Uesono Y; Toh-e A; Kikuchi A.
A new yeast gene, HTR1, required for growth at high temperature, is needed
for recovery from mating pheromone-induced G1 arrest.
Molecular and General Genetics, 1994 Oct 17, 245(1):107-16.

Nash R; Tokiwa G; Anand S; Erickson K; Futcher AB.
The WHI1+ gene of Saccharomyces cerevisiae tethers cell division to cell
size and is a cyclin homolog.
Embo Journal, 1988 Dec 20, 7(13):4335-46.

-- 
Kevin A. Morano
Dept. of Biological Chemistry
University of Michigan Medical School
Ann Arbor, MI 48109-0606



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