USF Health


Canto II User Guide

If the machine is not turned on.

You will need to turn on the machine approximately 30 min before using it to warm the lasers up properly.  Technically about 15 min is enough, but the industry standard is really 30. 

0)   If the computer is not on, turn it on and log on using the "User" icon.  No password is needed.

1)     Turn on the Canto II with the big green button on the left side.  Turn on the computer- you can do it in either order.  Log on the computer, and then double click on the FacsDiva icon to turn on the software.  Log in to the software.

2)     Look at the bottom of the “instrument” panel.  If it is not on the screen, click on the icon with a red star and a beam shooting out of it to the right in the upper left corner of the screen.  The bottom of the instrument panel will say if the instrument is connected or not.  If it is not connected, pick “connect” from the “instrument” link on top.

3)     Once the instrument is connected, click on “Fluidics Startup” from the top of the “instrument” menu. A dialog box may open asking if you want to proceed even though the HTS is not coupled.  If you are not using the high throughput mode click OK.  In the next dialog box again pick OK to start fluidics start up. This will take approximately 7 min.

If the machine is on

1b)  Make sure that it is under your user name, change user if necessary.

2b) Make sure that the instrument is connected, see 2)

3b) Make sure that fluidics is on: the bottom of the instrument panel should say “The system is ready”

Get the machine ready for acquisition

4)     Verify instrument configuration.  Choose Instrument> Instrument configuration.  For most works the 4-2 configuration is the correct one. Click Set if needed, then exit with clicking OK

5)     Create browser elements: under your folder create a new experiment and name it.

6)     Select experiment level instrument settings.  In the Inspector window click on the parameter tab and verify that only the parameters necessary for your experiment are listed.  Select the ones you don’t need and delete them.

7)     Apply initial PTM settings. The numbers you need are going to be  placed on the fluidics cart under the Canto II.

Compensation Controls

8)     Create compensation controls (unless it is a single color experiment).  Choose Instrument> Instrument setup > Create Compensation Controls  Unless you have several custom sets of dyes (you will know if you do), click OK.

9)     For the next step you will need a tube of unstained control and a tube that is positive for all your markers (if you don’t have one like that for sure, you can use your comp control tubes one by one here, too, it’s just more labor intense).  Put the pointer on the Browser on the unstained control tube and install the unstained control tube on the SIT.

10)  Hit Acquire on the Acquisition Dashboard at a Low flow rate.  Adjust FSC and SSC settings in Instrument window > Parameters so that your population of interest will fall in the neighborhood of 100-150 000 on both axis.

11) Choose Threshold tab in the Instrument window and adjust setting to exclude cellular debris.

12) Using the FSC/SCC dot plot adjust the P1 gate so that it would encompass your population of interest.  Right click on the border of this gate and choose Apply to Compensation Controls

13)  Adjust PTM values for all fluorochromes in Instrument window > Parameters so that the negative peaks are completely on scale.

14) Hit Stop acquiring and change the unstained control tube to one with positive staining.  Remember that the SIT will be flushed between samples, so let that happen before installing the next tube.  While keeping the arrow on the Unstained control hit Acquire. If necessary adjust PTM voltages so that the positive peaks are fully on scale.  Stop acquiring.

15)  Install Unstained control tube again with the pointer on the Unstained  Control tube in the Browser.  Hit Acquire.  Once the acquisition stabilized (perhaps 10 sec) hit Record Data in the Acquisition Dashboard.  Record all your controls.

16)  Check on the Worksheet window that the interval gate for each control encompasses the positive peaks.  If your positive controls are not-all-that positive, the important issue is to have the brightest cells in gate, not necessarily the dim ones.

17)  Choose Instrument> Instrument Setup> Calculate Compensation.  Name in the compensation setup in the new window, hit OK.

18)  Click on New Specimen button and rename it to something that when you are writing your paper 6 months from now will still mean something to you.

19)  Click on Tube_001 and add as many samples as you have, naming them.

20)  Choose Experiment> Experiment Layout, under the Labe tag enter appropriate labels for each tube

21)  Create on the Global Worksheet the appropriate worksheet elements for your analysis.

And finally here we go!

22)  Record your experimental tubes – Good Luck!

23)  Analyze your data

24)  Export your results

25)  Make sure that there is no other user shortly after you.  If there is, quit FacsDiva without shutting down the Canto II.

26)  Choose Instrument> Fluidics Shutdown and OK in the pop up window or windows if the HTS window would appear, too.

27)  Once the shutdowns is complete click OK and turn the Canto II off with the big green button on the left of the machine.