staining yeast for flow cytometry

Fatima Cvrckova FATIMA at AIMP.UNA.AC.AT
Fri Sep 30 05:26:26 EST 1994


Dear Colleen et al.,

this is what works for us. The procedure comes from Fred Cross and was 
developed to near perfection by Angelika Amon and Etienne Schwob.

Good luck!

Fatima

Harvest 5 ml of 10exp7 cells/ml sonicated for 10 sec and spin down. Resuspend 
in 2 ml 70% EtOH and transfer into a 2ml eppendorf tube. Place in a 23C rotor 
for 2 hr to overnight. 

Next morning pellet cells and wash twice with 1 ml 50mM TRIS pH7.8. Then 
resuspend in 0.8ml 50mM TRIS pH7.8 and add 20 µl of 10mg/ml RNAse (RNase should 
be preboiled for 20 min). Incubate on a rotor at 37C for at least 6 hours (I do 
over night).

Next morning pellet cells and resuspend in 0.5ml of 5mg/ml pepsin freshly 
disolved in 55mM HCl (5 µl /ml of the 37 % HCL). Incubate in a 37C water bath 
for 30 min. 

Spin down and wash once with 1ml of 200mM TRIS pH7.5, 211mM NaCl, 78mM MgCl2. 
Resuspend in 0.5ml of the above buffer and add 55 µl of 0.5mg/ml propidium 
iodide, thus the final concentration of the buffer is: 180mM TRIS, 190mM NaCl, 
70mM MgCl2.

At this stage, samples can be stored at -20 ºC.

Re-sonicate just before measuring.

Soon thereafter read on the FACS machine: Transfer 50 - 100 µl cells to 1 ml 
50mM TRIS pH7.8. Read immediately after dilution, read 10000 events per sample.



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