SITE SEARCH

Calibur start-up procedure
	1. Troubleshooting tip #1: Turn the cytometer on before the computer. They will not connect and you will not be able to connect to cytometer. If the computer is on and the cytometer is not, turn the cytometer off, then restart the cytometer, followed by the computer. 2. Open the drawer and check waste and supply tanks. If they require filling/emptying, release the pressure valve by pushing it into the back position. The waste may be dumped in the sink. Only the sheath will pressurize, so be sure the seal is hand-tight after filling. *Troubleshooting tip #2: Be sure the pressure switch is in the forward position. The Run button on the Calibur will be yellow instead of green if it is not.
FileGuard
	3. Log onto the computer by finding your username and entering your password. The total amount of time you are logged into the computer will be the amount of time you are billed. Troubleshooting tip #3: Your password is case sensitive, so check the caps lock if you get a message that your password is not valid.
Opening CellQuest from the Desktop
	4. Under the Apple symbol on the desktop, open CellQuest. If the desktop for CellQuest does not appear, go under File to New.
Establishing communication between the cytometer and the computer
	5. Under Acquire select Connect to Cytometer. If you can't select Connect to Cytometer, refer to troubleshooting tip #1. The Acquisition Control Panel should appear after the computer connects to thecytometer. If it does not, go under Windows and choose Acquisition Control Panel.
Creating a place to store Cellquest data
	6. Under Acquire, select Parameter Description. Select Folder by clicking on the gray box in the upper right-hand corner. You should select your ZIP disk to save your data to, as saving data to the hard drive is discouraged. If you are saving to the hard drive temporarily, select Desktop, FACS 1, Users, Your PI's name, your name (or new folder to create a file for yourself), and finally, New Folder. Name the folder you wish to save your data to. The date is the most common method of naming. Select this folder by clicking on the gray bar with the select (your folder). You now have a place to save your data.
Detectors and Amps and Instrument Settings
	7. Under Cytometer choose Detectors and Amps. Be sure the FSC and SSC are in linear, while the fluorescence parameters should be in log. Cell-cycle note: FL2 should be in linear, the DDM parameter at the bottom of the window should say FL2, and FL2-A should be selected instead of FL2-H. 8. If you have done this experiment before and wish to use the instrument settings already used, choose Cytometer, Instrument settings, and then find the file you want to pull the old instrument settingsfrom. When they have been found, click Set and Done to load them into the Detectors and Amps.
Choosing the Appropriate Plot
	9. Under Plots (or by using the tool bar) select Dot Plot. There will be a gray box that says Analysis. From the pull down menu that appears by clicking on Analysis, choose Acquisition?Analysis from the pull-down menu. The X-parameter should be FSC (forward scatter), which is the size of your cells. The Y-parameter should be SSC (side scatter), which shows the complexity and granularity of your cells. 10. Under Plots (or by using the tool bar) select the plot(s) appropriate to your experiment. The emission ranges for detection are as follows: FL1: 530 ± 15 FL2: 585 ± 21 FL3: 650-700 Cell cycle: use FL2-A Histograms: Use a histogram if you only want to look at one fluorochrome, or are doing cell-cycle analysis. Under Histogram choose Acquisition?Analysis as was done for the FSC and SSC dot plot. Use the pull down menu under the gray box around FSC-H to choose the appropriate parameter. Click OK. Dot Plots: Use a dot plot when you are using multiple fluorochromes. Choose Acquisition?Analysis as was done for the FSC and SSC dot plot. Use the pull-down menus under the X and Y parameters to chose the parameter appropriate for your fluorochromes. Click OK.
Putting a sample on the cytometer
	11. Place your first sample (negative control) on the SIP by swinging the arm out, gently placing your sample on the SIP, and swinging the arm back. Press Run on the front panel of the cytometer. Choose low, medium, or high depending upon the concentration of your cells. Check the Acquisition Control Panel to be sure the set-up box is checked then click Acquire.
Positioning the negative control on the FSC/SSC dot plot
	12. Using the Detectors and Amps, you will first adjust the FSC to center the cells on your FSC/SSC dot plot. E-1 is for very small cells; E00 is for larger cells,etc. Choose one of these so that the cells are not squished against the y-axis, and are also not lost off the end of the x-axis. Use the Amp gain to Adjust the cells to the center of the plot. Next, adjust the SSC by first using the voltage, then the Amp gain to center the cells by clicking on the arrow keys for up and down. Short cut: The center bar may be used to move quickly through the range of voltages, or the arrows will move by tens if the option key is held down when they are used.
Gating
	13. Using the palate, select a gate from the population of cells you want to look at on your FSC/SSC dot plot. Using the open apple key, grab one of the squares on the edge of the region to turn it to fit your population. These can also be used to expand or reduce the size of the region. If the region you have drawn is not correct, it can be deleted under Gates to Regions, where the region can be selected and then deleted using the Delete key. Under Plots, choose Format Histogram/dot plot. Change No Gate to G1=R1, or the name of the region you have drawn around your cells. If you can't decide from your Dot Plot which population is single-cells, open a Density Plot, which will show you where the greatest concentration of cells lies. These should be your single cells.
Positioning the negative control on the fluorescence plot
	14. Using the Detectors and Amps, you will next position your negative control in the 101 decade of your histogram, or in the 101 Decades of your dot plot (the lower left-hand corner) in the same way the voltage was adjusted for the FSC and SSC. 15. Close the Detectors and Amps window. You have established the background level of fluorescence and changing the voltage on any parameter will make your data null.
Compensation
	16. Multi-fluorochrome Experiments (single-fluorochrome experiments, skip to step #17): Compensation is used to remove the area of spectral overlap between FL1/FL2, FL2/FL3. Compensation is not used between FL1/FL3. First, select the Quadrant Marker from the Palate, placing the line at the 101 Decade on both the X and Y-axis. Under Acquire, select Compensation. Set all parameters to zero. Place you single-fluorochrome control on the SIP. The compensation will be increased to bring the signal under the quadrant marker towards the parameter (FL1, FL2, or FL3) for both single-fluorochrome controls. Remember, the parameter you are moving the signal towards should be on the right-hand side of the equation. For Example, If you were compensating a sample that fluoresces on FL1, you would choose FL2-FL1. Note: It is better to error on the side of too little compensation, as you don t want to lose double-positives by removing too much of the signal range. Close the Compensation window.
Setting the number of cells to be stored as a file
	17. The default setting for the number of cells collected and stored is 10,000. If this is not appropriate for your experiment, you may change the setting under the Acquire menu by selecting Acquisition and Storage.
Counters
	18. Under Acquire, select Counters. This gives the count of how many cells have been acquired.
Acquiring the data sets
	19. The first data set is now ready to be collected. Under Acquire, bring up the Parameter Description. Name your first sample under sample ID. On the Acquisition Control Panel, click Pause, Abort, uncheck the set-up box, and choose Acquire. The location of the folder the data will be saved to will appear on the Acquisition Control Panel. If the sample is taking too long to acquire the desired number of events, choose Pause on the Acquisition Control Panel, then Save. The computer will beep when it has saved your data file successfully. Optional: On the Parameter Description window, you may change the naming of the FL1 parameter by typing in the new name under P3, FL2 under p4, and FL3 under P5 (these correspond to the Detectors and Amps). Remove your first sample, place the next sample on the SIP, and click Acquire on the Acquisition Control Panel. Repeat for the rest of the samples.
Closing CellQuest after all data sets have been collected
	20. After all samples have been acquired, go to File, then to Quit. Choose Don't Save. An acquisition template can be created if you choose Save for use with future experiments.
Logging off FileGuard
	21. Log off of the computer by clicking on the shield in the upper right-hand corner, and scrolling down to Log Off.
Cleaning the machine
	22. Run 5 minutes of bleach and 5 minutes of water when you are done to reduce clogs. Be sure the machine is left on Standby with a tube of water on the SIP when you leave the facility. Turn off the computer and cytometer if you are the last user for the day.