staining yeast for flow cytometry
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.
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
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,
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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