From: David McFarland (David.McFarland@mcmail.vanderbilt.edu)
Date: Thu Jun 18 1998 - 17:11:09 EST
---------------------------- Forwarded with Changes ---------------------------
From: Ryoma Ohi at CELLBIO
Date: 6/18/98 4:08PM
To: cyto-inbox
Subject: FACS protocol
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Several list members asked me about a Sytox Green protocol. So here it is:
1. Spin down 1E7(yeast) cells in a microfuge for 1 minute.
2. Resuspend pellet in 1 ml of 70% EtOH. Fix for at least 60 minutes at room
temperature (or up to several days at 4 deg C).
3. Pellet cells and resuspend in 1 ml of 50 mM sodium citrate, pH 7.0
4. Sonicate (30% for 15 sec), pellet, and resuspend in 1 ml of the same
solution.
5. Add RNase A to 0.25 mg/ml. Incubate at 50 deg C for 1 hour or overnight
at 37 deg C.
6. Pellet and wash cells. Pellet again and resuspend in 1 ml of 50 mM sodium
citrate that contains 1 µM Sytox Green (stock is available from Molecular
Probes, S-7020, and is 5 mM). Keep at room temperature in the dark for at least
1 hour.
7. Analyze on flow cytometer.
This protocol is courtesy of Puck Ohi in the lab of Kathy Gould at Vanderbilt.
My two cents: Sytox green is very bright. The voltage/gain needed is about
half that of PI stained samples (of course it's also in a different channel,
hence the "Green"). It is also not base dependent/specific so cvs are much
better. Check the Molecular Probes catalogue for details. Keep the cell number
about the same in all tubes. The sonication step is about 90% technique. If
not done properly we get really weird results. Try not to whip air into the
buffer. By the way, they don't use a water-bath style sonicator. It is a probe
that slides down into the tube, but don't touch the sides of the tube! Kathy
likes to sonicate in 1.5 ml microfuge tubes and then transfer to the flow tubes.
David McFarland
Howard Hughes Medical Institute
Flow Cytometry Facility
Vanderbilt University Medical Center
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