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FACScan OPERATION

Please note that the following is a summary of how to operate the BD FACScan and does not take the place of hands-on training.

Only use BD Falcon O.D.. x L: 12 x 75mm Tubes (No.:352008, Fisher No.: 14-959-5†, A case of 1000 for $53.60) for BD flow cytometers

START-UP PROCEDURE

FACScan
                

  • Turn on the FACScan by flipping the switch on the upper left corner of the front instrument panel. Open the bottom panel door and check the waste (right) and sheath fluid (left) containers. If sheath fluid is low or empty unscrew the cap to release the air pressure. Let the sheath fluid sit like that while you unscrew the cap on the waste container. Remove the waste container and empty into the sink. Return the waste container to the instrument and recap. Pull out the sheath container and use the bottles of saline provided (0.9% NaCl ) to refill sheath container. Make sure you slide the sheath container back into the instrument BEFORE you tighten the cap. Make sure the cap makes a tight seal or the vacuum will not work.
  • Turn on the computer. The FACScan and computer will take a few minutes before you are ready to collect data. Usually this start-up procedure is done by the management at the beginning of each day so you won't have to worry with it. Also, quality control is performed each day to insure that the instrument is functioning properly.

 

  • The indicator light on the FACScan will read "Not Ready" until the laser is properly charged. The fluid control knob should be in the "Standby" position. When the indicator light changes to "Standby" the FACScan is ready to acquire data.

 

ACQUIRING DATA

  • First, open the "Scan User Folder" by double clicking on it. Double click on "Scan Data folder" to open. Go to "Acquisition,Templates" and find a template that will best fit your samples. For example, if you only have FITC in your samples, pick the "FITC panel" and double click to open it.

 

  • When you open the template you will automatically launch CellQuest, the software used in acquiring data. From the pull down menu at the top of the screen choose "Acquire" and go to "Connect to the Cytometer". An acquisition box will appear. Check the "Setup" box.

 

  • Go to "Cytometer" on the pull down menu and choose the "Detectors", "Compensation" and "Threshold" panels. These are your acquisition tools.
  • Next create a data file to hold your data. Go to “Acquire" on the pull down menu and select "Parameter Description". A large box will appear. In the upper right hand corner click on "Folder". You will be given a choice as to where to store your data. We have created a folder called "Scan Data Folders". Inside this folder is the name of all the PI's that use this facility. Choose your PI and then click "New". Name your file using today's date your initials to identify your experiment. Click "Select".

 

  • Now click on "File". Name your file by using the same name as your folder, making sure the counter is on "1". Click "OK". This will be your tube number.

 

  • Set flow rate at Low (~20 μl/min) when setting parameters and High (~50 μl/min) when acquire data from samples.
  • Turn the fluid control knob to "Run". The indicator light should change from "Standby" to "Ready". You are now ready to put on a control tube, meaning a tube containing NO stain OR isotype staining only. This will allow you to set the PMT voltages and compensation for baseline or negativity.

 

  • Place the tube on the sample port nozzle and swing the arm underneath. This allows the vacuum to be created in the tube and start the sample flowing through the instrument. Click "Acquire" in the acquisition box. Within about 5 seconds you should see data on the screen. The cells, or "events" are represented by dots on the screen.
  • First, adjust the Forward and Side Scatter voltages to place your cells in the upper left dot plot. Forward scatter is a measure of the size of your cells and Side scatter is a measure of the internal complexity of your cells. The voltage and amp gain are cell dependent. Increasing the FSC value moves the cell population from left to right; decreasing FSC moves the population from right to left. Increasing the SSC value moves the cell population up; decreasing the SSC moves the population down. Keep adjusting until your population is positioned in the dot plot inside the gate or wherever you want it. You can also move the gate around by double clicking on it and dragging it around with the mouse. Now you can move on to the FL channels.

 

  • Adjust the PMT Voltages (located on the "Detectors" panel) so that the peak fluorescence of your control tube is between 100 and 101 on a histogram plot. What fluorchrome you are using will dictate which PMT's you will have to adjust. For example, FL1=FITC, FL2=PE, FL3=PerCP, and FL4=APC. Increasing the value will move the population from left to right; decreasing will move it from right to left. Once you have the PMT's set it is time to move to Compensation.
  • Remove the negative control tube and replace it with a tube containing a known positive for the fluorochrome you are using. Adjust the compensation using the "Compensation" control panel. This will take a little practice so don't get discouraged! During the tutorial we will explain how to compensate your data. Just ask Enid, Tracey or Marion for help anytime. Once you have the compensation set you are ready to acquire the rest of your samples!

 

  • Go to the Acquisition box and deselect the Setup mode by clicking in the box by Setup. Go to the "Acquire" pull down menu and select "Acquisition and Storage". A window will appear allowing you to set your acquisition requirements such as parameters saved and how many events you want to acquire. For most of the time 10,000 events are sufficient. When you have everything set like you want click "OK". Go back to the "Acquire" menu and select "Counters". To open the box fully click once in the tiny box in the upper right hand corner. Click on the button that says "Rate" and change it to "Accumulate". This will show you how fast your data is running and let you know when you have acquired your specified number of events.
  • Place tube #1 on the sample port and click "Acquire" in the acquisition box. The instrument will automatically acquire the specified number of events and stop when done. The computer will "beep". Take off that tube and proceed with tube #2 and so on until you have acquired all of your tubes. You do not have to rinse between samples. When you have finished place a tube of deionized water on the sample port and run for 3-4 minutes UNLESS you have just used PI in the system. If PI was used place a tube of bleach on first, run for 3-4 minutes then place the water on and run for another 3-4 minutes. Turn the fluid dial to "Standby". NEVER WALK AWAY AND LEAVE THE INSTRUMENT IN THE "RUN" POSITION!! You are now ready to analyze your data.

 

DATA ANALYSIS

  • DO NOT CLOSE CELLQUEST!! You can acquire and analyze in the same template!

 

  • Using the mouse place the arrow on the white background of your template page and click once.
  • Go to the "EDIT" menu; click and go to "Select AII”. Your dot plots/histograms should have the corners highlighted with little black squares.

 

  • Go to the "PLOTS" menu; click and go to "Change Data File". A new window will open allowing you to navigate to your data folder. Under "Open a Data File" use this button to navigate to your folder that is named for your PI. Highlight your PI's name, click "Open". Highlight the folder that you created for your experiment and click "Open". The highlighter should already be on your first file, or tube number. Click "Open". Your data should be visible in the template now. The file number, or tube number, will be at the top of the graphs.
  • To scroll through your data tube by tube go to the "PLOTS" menu and choose "Next Data File”. This will advance to the very next tube and so on. You can also go backwards by choosing "Previous Data File" from the "PLOTS" menu.

 

  • Gates, Regions, markers and quadrants can be moved by simply placing the mouse arrow on the line and double clicking on it. You now have "handles" you can grab with the mouse arrow by clicking on one of the little black squares, hold down the mouse button and drag the line where you want it to go and let go of the mouse button. Markers can be moved by using the mouse to click on the end of the marker bar, hold it down and move it to the left or right as desired. Click on the middle of the quadrant marker where the two lines intersect and set it using your control tubes.
  • Once you are satisfied with your gate, region, marker, and quadrant placements click on the white background again and go to the "EDIT" menu and choose "Select All" to activate the plots.

 

  • Go to the "PLOTS" menu and choose "Change Data File". Once again, use the window to navigate to the first sample in your file folder.
  • Once you have chosen your first file number, go to the "BATCH" menu, and choose "Setup". In the window that appears make sure the "Print after each file increment" has a check mark in the box. If not, check this box. This will tell the computer to print after each file.

 

  • Go back to the "BATCH" menu and select "Run". The computer will spool all of your data to be printed. When done it will tell you that the end of the file has been reached. Click "OK" and your data will begin printing.
  • When all of your files have been printed, close CellQuest by going to the "FILE" menu and selecting "Quit". In the window that pops up select "Don't save". This will close out CellQuest.

 

    • VERY IMPORTANT!!! Again, please make sure you leave the instrument in the "Standby" position before walking out of the lab. If you need special gating or help with setting markers or regions just ask Enid or Tracey for help! Please feel free to make suggestions or comments concerning this tutorial handout. Your input is very important to us! Thanks.