This protocol is for FL4 setup for the FACS calibur.

We use the molecular probes 633 6 µ beads A7313. Dilute 7 drops in about 4 mls of sheath buffer. To make the template for the calibration all you need is 2 histograms: one for FSC and one for FL4. You need a marker on both and a stats box so you can monitor the mean fluorescence intensity.

Instrument settings

• Put the threshold on FSC and set to 200.
• Adjust FSC so the peak is about 400 and adjust the FL4 voltage until the peak is about 800.
• Save a data file to the same folder as your template for future use.
• The time delay calibration for the FACS calibur should be run every time you use the instrument. The calibration is dependent on the sheath pressure, which can vary slightly each time the tank is pressurized.
• First make sure the cap on the sheath tank is screwed on firmly. Don't try to screw it on super tight or the cap will break.

Procedure for setting up the time delay

• Find the 12X75 mm tube in the fridge called Align flow 6µ beads.
• Locate the FL4 setup folder on the desktop.
• Open the template 'Time Delay Calibration'.
• Recover the instrument settings from the Alignflow 6u.001 data file. It should be in the same folder.
• Place the tube on the SIP.
• With acquisition in setup mode and sample pressure on medium, adjust the FSC gain to put the peak at channel 400,+/- 5.
• Adjust the FL4 PMT voltage so that the peakis at channel 800. If it is far from that channel, something is wrong. Repressurize the sheath tank and ensure the cap is tight.
• Select 'time delay calibration' from the cytometer menu and click on calibrate.

If there is no error message, the machine has calibrated correctly. If the instrument does give an error message, such as 'signal out of range' go to Simon. If he's not arround, depressurize and repressurize the sheath tank and try again.