SIMPLIFIED INSTRUCTIONS FOR USERS OF THE SKATRON ARGUS FLOW CYTOMETER

BEFORE SWITCHING ON

1. Read these instructions.
2. Consult the "Aber Flow Cytometry: guidelines for sample preparation" sheet
3. Check that the sheath container has enough fluid.
   • Open the lower left door (labelled SHEATH)
   • Check the level in the tank. If it is less than ¼ full you are advised to refill it
   • Disconnect the metal level sensor plug with the black lead.
   • Disconnect the plastic drain tube.
   • Slide out the tank and remove the grey plastic lid.
   • Add sheath fluid prepared using 1mM Sodium azide in water that has been filtered through a 0.2 mm filter. Millipore water can be obtained from the preparation room (next to T34) located opposite the Skatron. Follow the instructions on the Millipore apparatus carefully.
   • Seal the tank and slide it back into the instrument and reconnect the sensor and drain tube.
4. Check to see if the waste tank needs emptying.
   • Open the lower right door (labelled WASTE)
   • Check the level in the tank. If it is more than ¾ full you are advised to empty it.
   • Disconnect the sensor and drain tube and remove the tank as described for the Sheath tank above.
   • Dispose of the waste liquid safely.
   • Replace the tank and reconnect the sensor and drain tube.

SWITCHING ON

1. Switch on the computer screen.
2. Switch on the switch at the rear-right of the instrument.
3. Switch on the lamp.
4. Switch the sheath flow on.
5. Switch the sample to AUTO mode.
6. Type SKAT on the computer and press Enter.
7. Select USER with the cursor keys and change the user to your username
8. Select MEASURE and press Enter.

ALIGNING THE FLOW CYTOMETER

1. Place a waste container below the sample point and press FLUSH. Wait for the flush cycle to end.
2. Choose your alignment sample (this can be beads if you have bought some) or cells whose flow cytometric properties you are familiar with).
3. Place the sample at a concentration of approximately 105 to 107 particles/ml on the sample point and press RUN.
4. When the "INFO 001" message flashes on the Sample window press HIGHER until the sample speed is set at 20 or 50 ml.min-1.
5. Open the top section of the flow cytometer.
6. Look down the eyepiece on the left hand side of the instrument while holding in the spring-loaded knob below it.
7. If the instrument is aligned you should see your sample passing through the instrument (a series of horizontal lines).
8. If you can't see anything wait for a few seconds and look again.
9. If you still can't see any lines increase the sample speed by pressing HIGHER
10. If you still haven't seen your sample check that the RUN light is still illuminated. If not go back to step 3.
11. If the RUN light is illuminated check that the nozzle is touching the cover slip and that liquid is coming out of it.
12. If you still can't see any lines turn the knob with the brass ring (1) to move the sample into view. IF YOU STILL DON'T SEE ANY LINES ASK FOR HELP.
13. When the sample is in view use the small focus knob (2) if necessary to sharply focus the lines.
14. Remove your alignment sample from the stage.
15. Press RUN to extinguish the light on the RUN button.
16. Place a waste container below the sample point and press FLUSH. Wait for the flush cycle to end.

RUNNING SAMPLES

1. Place your first sample on the stage and press RUN.
2. When the "INFO 001" message flashes on the Sample window set the sample speed to 20 ml.min-1.
3. Make sure that the settings on the computer are the ones that you want to use.
4. Enter the identity of your sample at the bottom left of the screen.
5. Press PAGE DOWN to move to the graph screen.
6. Press control-F3 to delete the data from the display.
7. Press F2 to start data acquisition.
8. Adjust the sample flow rate to give a sensible data acquisition rate (between 10 and 100 counts.sec-1.
9. Press control-F3 to restart acquisition.
10. Acquire your data then press F2 to stop acquisition.
11. Press F4 to save your data and note the file name.
12. Remove your sample from the stage.
13. Always use the FLUSH cycle between samples.
14. Run the remainder of your samples using steps 1-12.
15. Always run the FLUSH cycle when you have finished.

BEFORE YOU LEAVE

1. Ensure that you have removed your final sample from the sample stage.
2. Ensure that you have run the flush cycle.
3. Switch off the Sheath flow.
4. Switch off the lamp.
5. Switch off the flow cytometer at the rear-right of the instrument.
6. Press END on the computer and select Y and then EXIT-EXIT to DOS.
7. Backup all of your data files onto a floppy disk.
8. Switch off the computer screen. DO NOT SWITCH OFF THE PC.
9. Clear away any mess, wipe up any spills and take all of your sample tubes etc. away with you.