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FACS Scan DNA content analysis

This protocol is for DNA content analysis using Arabidopsis nuclei.

First, reserve time on the Optical Biology website, or get Simon to do this for you. If you haven't used the machine before, you'll need to be trained by Carol Oxford or by someone in the lab. Then make your nuclei one day ahead so you can stain them overnight with propidium iodide.

1) Set up the FACS Scan and computer.

• Turn FACS Scan on, power button is at the left. The FACS Scan must be on before the computer, so restart the computer if necessary.
  
• Make sure that sheath and waste tanks are connected correctly and that the sheath contains some liquid. If you need to change these, switch off the alarm on the pump first. The old sheath container becomes the waste.
• Make sure the Cytek pump is on. If the filter is filled with air, allow the pump to fill it with sheath. Open the front panel on the FACS Scan to check this.
• Open the Cellquest application. Acquire > Connect to cytometer.

2) Set the parameters for data collection.

• Use the FCS (flow cytometry standard) file from the Comai lab. This is in the Cellquest file.
• Acquire > Parameter Description. Name your data file using the following format SC mmddyyy e.g. SC 0511007
• Setup mode

3) Adjust the thresholds for data collection.

• Put a tube into the machine, and set the fluid control switch on the machine to low.
• The FL2 laser is for propidium iodide.
• You want peak area x peak width to be linear. Area (or height) = amount of fluorescence, Width = time i.e. want one nucleus, not two.
• Change the threshold for each until the graph looks good.
• Ideally, the diploid peak should be at 200 x 200

4) Collect data.

• Unclick Setup to acquire.
• Change the # of events in Acquire > Acquisition and Storage.
• Check on the flow rate. Flick tube if the nuclei are too sticky, or change the fluid control switch to high (low is best for accuracy).
• Pause > Save can stop the sample, after you have chosen to acquire.
• Change Stats > Histogram Stats will show the data

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