This page is currently under construction - a full SOP for operating the FACSCalibur, Loader and MAS will follow soon!
There is now only one shiny, almost new FACSCalibur (6 parameter, dual laser, but no sorting) with permanenetly attached FACSLoader (for automated running of up to 48 samples per rack in tubes) and Multiwell AutoSampler (MAS: for automated running from 96 well plates). Bookings are made on the form on the front of the RED FILE that also contains a log of all runs and copies of Standard Operating Procedures. The FACSort has now moved to the Dunn School Central Flow Cytometry facility run by Nigel Rust. The old FACScan and FACSMate will very soon be decomissioned as the 96 well plate facility is available on the FACSCalibur (which runs plates roughly twice as fast as the FACSMate).
All samples must be fixed (2% formalin) for use on this machine - for live cells (or use of propidium iodide) please use the machines in the central facility run by Nigel Rust.
NB. Main instructions are in BOLD FACE, with subsidiary information in normal type.
Check that the FACSFlow Supply System (under the bench) is switched on and that no alarms (indicating either an empty sheath or a full waste container) are either sounding or show a warning light. If the waste container (on right) is either showing as full or looks almost full then carefully remove the screw top, empty the containiner down the sink and then reattach (depress the waste container top down after reattaching to ensure no pressure build-up and to ensure waste full sensor in the cap extends into the container). If the sheath container (on left) indicates less than 1.5 litres remaining then unscrew the cap (be careful not to damage the sensors) and fill it up with sheath fluid (filtered deionised water from the tall, grey taps in room 47/48 (214.20.36), plus 0.1% w/v sodium azide). Then press the RESET button to cancel any alarms. Ensure that the sheath supply tubing from the container is not kinked on either side of the inline filter.
B) Setting up the Sample Input Port (and Cytometer Interface Unit)
It is best to change the Sample Input Port (SIP) setup before switching on the FACSCalibur machine. Before changing the SIP setup ensure the sheath tank in the FACSCalibur itself is depressurized (toggle switch between the tanks in the sliding shelf). The SIP should normally be left with a tube of water held in place by the lower tube support arm to stop it drying out - the support arm should be moved to the side and the tube removed before proceeding with setting up the SIP further.
There are three basic arrangements for the Sample Input Port (see diagram below).
(1) Manual
The first setup is for manual, single tube operation. In this case the Cytometer Interface Unit (CIU) is not required and should be parked in the top left hand back corner of the sampling area using the inbuilt magnets. The transparent tube guide is also not required and if present can be removed and replaced with the standard black metal washer. The vacuum sleeve (or droplet containment module: DCM) should be screwed in place ensuring that both rubber O-rings (one in the DCM and one to seal with the sample tube) are in place. Once a sample tube is in place the lower tube support arm should be moved in place to run the sample (adjusting the height to support the tube if necessary).
(2) Automatic or Manual Operation of Tube Loader (DEFAULT SETUP-the machine should be left like this!)
This setup is for using the FACSLoader with the circular 40 tube racks. Again, the CIU is not required and should be parked in the top left hand back corner of the sampling area using the inbuilt magnets. Ensure that the black rubber O-Ring that normally makes a seal with the sample tube is removed (the tube seal is made instead with the orange washer at the top of the SIP although this is not visible with the tube guide installed). The transparent tube guide should be installed, replacing the standard black washer (it unscrews) at the top of the sample port. The lower tube support arm should be pushed sideways to allow tubes to move freely up and down between the loader rack and the SIP.
(3) Automatic Operation from Multiwell AutoSampler (MAS)
The third setup is for autosampling from 96 well plates via the MAS and Cytometer Interface Unit (CIU). The transparent tube guide is not required and if present can be removed and replaced with the standard black metal washer. The vacuum sleeve (droplet containment module or DCM) should be removed (including the small black rubber O-ring inside it), but the larger black O-ring that seals with the sample tubes (or the CIU) should remain in place. Finally, the CIU should be taken from the parked position (held by magnets) and placed in position as if it were a sample tube, with the lower tube support arm placed underneath (this may need a slight adjustment to the height by screwing the support up or down).
Please place the parts that are not used in the chosen setup in the plastic bag clipped to the side of the machine.
Please ensure that the fine tubes attached to the CIU are not pulled, kinked or bent, as they are rather fragile and any damage will stop reliable operation of the MAS.
C) Switching on the FACSCalibur and other equipment
First ensure that the Macintosh computer attached to the machine is shut down. Then switch on the FACSCalibur (green button on right hand side of instrument, followed by the MAS if required (green button front left), followed by the manual keypad (rocker switch on pad), and lastly, (re)start the Macintosh computer (round button top right of keyboard).
Ensure the toggle switch between the sheath and waste tanks in the FACSCalibur is in the Pressurised - RUN position, and that there are no air bubbles in the sheath filter (if there are bubbles these can be bled through to the waste tank by releasing the sliding clip then closing it again). It is also a good idea to check that the small sheath tank in the FACSCalibur is not completely empty (especially if somebody let the large tank on the FACSFlow system run dry!) and that the waste tank is not completely full (if so this is probably a kinked tubing running down to the FACSFlow waste).
D) Preparing the FACSCalibur and CellQuest for aquisition
This page last updated by Steve Cobbold 18th March 2002.