Sean Lauber:Using the BDCanto Flow Cytometer

Using the BDCanto

Machine needs to be turned on:

1. Turn on machine using green button on left of machine

2. Check all the levels of the fluids below the machine

 Check by weight or eye
 If the sheath buffer or other buffer needs filling, fill a graduated cylinder with sheath and pour this into container (check under sink)
 If need to fill with distilled water, disconnect the unit (push-click one of them, simply pull the other one) and fill with distilled water in sink. Or use a squirt bottle.
 If need to empty waste (full or near 10 L), disconnect and dump in sink (pull cap off – don’t unscrew) (let water run 3 min) then fill with 1 L bleach
 To reconnect the wires, line up the arrow and push in for one of them. For the other just push-click it.

3. Login to computer

4. Let machine warm up for 30 minutes

5. Run Fluidics startup (cytometer/fluidics startup)

6. Degas the machine to clear bubbles (cytometer/cleaning mode/degas)

7. Load a water sample and run through machine for two minutes (acquire data, collect at HIGH)

8. Proceed to compensation

Machine already turned on:

1. Ensure green button on left of machine is on

2. Check levels as indicated above

3. Login to computer

4. Prime machine (cytometer/cleaning mode/prime)

5. Load a water sample and run through machine for 2 minutes (acquire data)

Compensation

1. Highlight the yellow folder

2. Create a new experiment

3. Highlight the experiment and create a new specimen

4. Press the acquisition control pointer on any tube and go to the Cytometer window. Go to the Parameters tab and delete any colors you don’t need (max=8). Select “W” for FSC and SSC to collect data for these parameters.

5. Select Experiment/Create Compensation Tubes

 New specimen with compensation tubes appear along with plots in the worksheet
 Create grids for each compensation plot

6. Select the acquisition pointer in front of the Unstained Tube

 Load the unstained sample into the machine and acquire data
 Adjust the Parameters/FSC and SSC voltage (in Cytometer window) in order to capture all events – RECORD YOUR VOLTAGES 
 Adjust the voltages for each stain to put the negative peak in the middle of the first quadrant, and coming down to end halfway into the 2nd quadrant
 Stop acquiring and remove the unstained sample (Remove Tube – during this time you must remove the tube quickly and snap back the receiving arm to trigger the wash)

7. Click next tube and the acquisition pointer will go to the next sample (first stained comp bead)

 Load your first stained comp bead sample, acquire data
 Using the plots in the worksheet area, make sure the positive signal is between 104-105. If it isn’t, adjust the voltage in Parameters to make it fall in this region. RECORD YOUR VOLTAGES.
 Stop acquiring and go to the next comp bead sample

8. If at any point you need to change one of the voltages of a previous compensation, you need to go back and change that voltage for those tubes before recording. When you start recording you MUST have all the voltages the same for each tube.

9. Now that you have all your voltages you need to record the events (5000 events) to calculate compensation

 First load the unstained comp bead sample, acquire for 5 seconds, then click record. Ensure (using worksheet plots) that everything looks okay
 Then record the next comp bead sample…After recording, the P2 gate should snap to the positive peak, if it doesn’t then try recording again. Remember it must be below 10^5

10. To calculate compensation go to Experiment/Instrument Setup/Calculate Compensation

11. Go to the Cytometer window and check that the spectral overlap for compensation doesn’t exceed 100

12. Link and Save

Loading samples

1. Now that you‘ve setup compensation, select your specimen outside of the compensation specimen and click the acquisition pointer on the tube in that specimen. Go to the acquisition dashboard and adjust the parameters you’d like for collecting events

 Usually you want to collect 10,000 events (display 1000). If you have few cells you might want to collect 50,000

2. Now begin clicking “Next Tube” until you have added many tubes as you’ll need

3. Create the plots

 In the worksheet, click “global worksheet” and begin drawing plots. Make as many colors as you have plus one.
   The first plot should be SSC-A (y-axis vs FSC-A (x-axis)
   The next plot will be SSC-A vs color 1, then color 2, etc.

4. Next load your sample into the machine and acquire for 5 seconds (ensure that events are being detected) before clicking Record. Try to collect about 2000 events/second, adjust the flow rate (low, medium, high) to achieve this. Once all the events are collected, Remove Tube (and then remove tube and snap arm back), then click Next Tube to go to the next sample…

Exporting Data

1. Once you‘re done, right click on the experiment folder you’ve recorded your data in and Export FCS. Make sure it’s in the FCS 3.0 format and go for it. Save it to your folder, and then use a USB to collect the data and delete it.