GUIDELINES FOR USING THE COULTER EPICS ELITE FLOW CYTOMETER IN IBS, ABERYSTWYTH

NOTE: Only authorised users are allowed to use this instrument. The current list of authorised users is limited to Hazel Davey (all procedures) and Kath Law (running samples and partial alignment only). No user should attempt any procedure that they do not feel comfortable with and these instructions should be used only as an accessory to the Coulter manuals located next to the instrument. If in doubt contact Hazel (Aberystwyth users only please!!). These instructions are also provided for the information of people who provide samples for us to analyse; i.e. to give them information on how the instrument is operated and configured. They are only applicable to the instrument at Aberystwyth. Use by others is entirely at their own risk.

BEFORE SWITCHING ON

  1. Read these instructions.
  2. Check there is enough free disk space on the C drive. (Type DIR, there should be at least 20MB free).
  3. Consult the "Aber Flow Cytometry: guidelines for sample preparation" sheet

SWITCHING ON

  1. Fill the Sheath tank and empty the Waste tank. Check that the distilled water wash has sufficient water.
  2. Ensure that the PC, 2 flow cytometer power leads and, if required, the HeCd and Green HeNe laser power leads are all connected to the mains.
  3. Switch on the MAIN POWER switch on the flow cytometer. (If there is a "grinding noise" switch off, wait a few seconds and try again!)
  4. Ensure that the AUXILIARY POWER switch is on.
  5. Type CYTOMETE on the PC. Select APPLICATIONS-UTILITIES and select your personal configuration. Select APPLICATIONS-ACQUISITION.
  6. Turn on the laser(s) using the touch screen. Laser 1 is the blue Argon Laser, Laser 2 is the red HeNe Laser. The Green HeNe laser is turned on using the key on the box, The HeCd laser is turned on by switching the knob from SHUTDOWN to OPERATE.
  7. Press the POWER UP VALVES button on the touch screen. Wait for valve actions to complete.
  8. On the PC select an appropriate protocol file. In the functions box make sure that CytoSettings is set to "send". Press F9 to send the settings to the flow cytometer. IMPORTANT: Allow 30 minutes for the instrument to warm-up.

ACQUIRING DATA

  1. Check the alignment of the flow cytometer using an appropriate protocol and sample - e.g. the "Immunocheck Hazel May 94" protocol and Immunocheck beads.
  2. If necessary carry out a partial or full alignment.
  3. Run the samples by pressing F9 to start and F10 to stop.
  4. Press F12 to enter sample information and an appropriate filename.
  5. Choose FILE-SAVE from the Histogram display screen to save the data. You are recommended to use the LISTMODE option as it gives the most flexibility for data analysis. The HISTOGRAM option is useful for overlaying samples within the Coulter software

SYSTEM SHUTDOWN

  1. Run a sample of COULTER CLENZ or 5% bleach for 3-5 minutes.
  2. Use the touch screen to turn the lasers off. If the Green HeNe laser has been used switch it off using the key. If the HeCd laser has been used switch it from OPERATE to SHUTDOWN AND WAIT FOR ALL LIGHTS TO BE EXTINGUISHED BEFORE PROCEEDING.
  3. Press SHUTDOWN VALVES on the touch screen. Wait for the valve actions to be completed.
  4. Wait for a further 2 minutes before switching off the MAIN POWER switch on the flow cytometer.
  5. Quit the flow cytometer software on the PC by choosing APPLICATIONS-EXIT.

BEFORE YOU LEAVE

  1. Dry the sample tube area and analysis area and any other spills with a tissue. Dispose of any hazardous waste in the sample tubes and waste tank appropriately
  2. Backup all of your data files over the network or onto a floppy disk.
  3. Unplug the pc.

ALIGNMENT PROCEDURE

  1. FOR FULL ALIGNMENT- START HERE

  2. Press VACUUM.
  3. Insert laser interlock.
  4. Remove beam-shaper.
  5. Remove, clean and replace flow cell if necessary.
  6. Loosen knob on top of flow chamber and rotate the flow cell so that the laser beam reflects back on itself. With the Ar laser look for the blue dot and send it back down the slit. Re-tighten knob.
  7. Replace beam-shaper.
  8. Select OPTIONS screen and toggle ALIGN LED to continuous. Select MAIN.
  9. Insert mirror into first filter slot.
  10. Close laser shutter. alignment knobs
  11. Adjust flow cell vert. (see diagram) and flow cell horiz. to centre LED image in the parabolic mirror.
  12. DISPLAY CAMERA and adjust it fully UP, ZOOM OUT and PAN RIGHT. Adjust flow cell vert. and flow cell horiz. to get the brightest edges of the flow cell.
  13. Select BEAM CENTRE and move the cursor fast right to bisect the image.
  14. Turn off ALIGN LED and select MAIN. Remove mirror from filter slot and press SHEATH.
  15. Open laser shutter.
  16. Adjust beam vert. until beam appears to pass through the centre of the parabolic mirror.
  17. Adjust beam vert. until the laser image intersects the cursor on the camera screen.
  18. Select the SCOPE screen and switch on the scope.
  19. Select TRACE 1 (bottom) and choose FS INT.
  20. Select TRACE 2 (top) and choose PMT2 PEAK.
  21. RUN a sample of immunocheck beads.
  22. Adjust beam horiz. until beam is roughly in the centre of the beam absorber in front of the FS detector.
  23. Remove Laser Interlock and close inner door.

    FOR PARTIAL ALIGNMENT- START HERE

  24. Adjust beam horiz. for maximum FS pulse height.
  25. Adjust PMT2 HVOLT for a PMT2 pulse of about 5 volts high.
  26. Select SCOPE, set TRACE 1 to be PMT2 PEAK and TRACE 2 to be PMT3 PEAK.
  27. Select MAIN and adjust PMT3 HVOLT for a PMT3 pulse of about 5 volts high.
  28. Adjust flow cell horiz., flow cell vert. and beam vert. for maximum PMT2 pulse height. If necessary reduce PMT HVOLT's to keep signal on scale. Repeat this step.
  29. Adjust fluorescence pickup lens (in filter chamber) for maximum pulse heights.
  30. Setup a graph of FS versus PMT1. Adjust the beam horiz. for best image (high, right and tight!)

SHEATH FLUID PREPARATION

The sheath fluid use in the Aberystwyth Coulter Epics Elite contains: Adjust to pH 6.8 with conc. KOH

To prepare 20 litres of sheath fluid:

  1. Dissolve 223.65 g KCl + 47.66 g Hepes in 1 litre of Millipore-MilliQ treated water (0.22 mm filtered) and adjust the pH to 6.8.
  2. Filter the prepared sheath fluid through a 0.1 mm filter into the storage container.
  3. Store at 4oC.
  4. When required dilute 1 part stock with 19 parts fresh MilliQ water.

DISTILLED WATER WASH REFILL

When required the distilled water wash bottle is refilled using Millipore-MilliQ treated water.

HomeFlow Cytometry Home Page

Author: Hazel Davey

[Mail Me] hlr@aber.ac.uk