How to use the Cytoflex LX
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Setting up an experiment (tubes)
Selecting the Proper Sample Injection Mode
Running the System Startup Program
Saving the experiment and exporting FCS files (tubes)
Setting up an experiment (plates)
Selecting the Proper Sample Injection Mode
Running the System Startup Program
Start the Cytoflex LX
Turn on the computer:
User: cytoflex LX
Password: password
Check if you need to change the sheath and/or waste
How to change sheath fluid and waste tanks
1. Waste tank
Check if the waste tank is full. If so, empty it according to the following procedure. Please wear PPE. Unscrew the green line coming from waste tank and leave it in a safe place with the probe facing the opposite side from you. Transfer the tank to the sink and empty it very carefully wearing gloves, lab coat and goggles. In the empty tank put 3 Virkon tablets and screw the cap back.
2. Sheath fluid tank
Check if the sheath fluid tank is empty or not enough for your experiment. If so, top it up with distilled water or replace it with a full one (under the bench). To do this, unscrew the lid, place it in a safe place – avoid touching the surfaces – and transfer the tank to the sink where the distilled water unit is. Top up with water and put the full tank back in place.
Setting up an experiment (tubes)
Set up the experiment in CytExpert software
Open CytExpert software (CytExpert desktop icon).
The Start-up screen appears.
To turn on the cytometer go to ‘Cytometer’ tab and select ‘Turn on’
Verify that the connection indicator light in the lower left corner of the software screen is green, and Connected is displayed. The left side shows the connection status, the middle shows the instrument status, and the right side shows the status details. If you see red indicators, see ‘Troubleshooting’.
Verify that the Sheath and Waste flow indicators in the lower right corner of the software screen are green indicating that the fluidics system is normal.
Selecting the Proper Sample Injection Mode
To run sample in tubes, follow these steps:
Go to Cytometer > Sample Injection mode > Semi automatic.
The following message will appear. Press ’OK’ and turn off the cytometer (Cytometer > Turn off). Then, turn the cytometer back on (Cytometer > Turn on).
Check the metal thing above the sample holder is on tube mode (see picture). If it is not, twist it the way it shows ‘T’.
Running the System Startup Program
If you are the first person using the instrument in the morning, you should perform ‘System Startup program’.
Go to Cytometer > System Start up Program
The following message will appear. Press ‘Initialise’ – the instrument goes to sleep mode if it is not used for 3 mins. The sample tube holder automatically shifts into the sample loading position.
Fill a clean tube with 2mLs of deionised water and place it into the tube holder. Press ‘Start’. The procedure takes approx. 10 mins.
When the procedure is finished, press ‘Close’.
Creating an experiment
Follow one of the 3 following methods:
1. To create a new experiment:
Select New Experiment
Specify the file path, and save the experiment. Desktop > data > 2022 > create a folder with your name. This is only a temporary folder. Please remember to take your data after finishing the experiment. This is very important, in case something happened to the computer you might lose your data.
Go to ‘Settings’ tab > Set Channel. All the available laser-channels appear in a window. Deselect the ones you are not going to use. Click ‘Close’. This will apply to every tube you are going to create or have already created. If you click ‘Apply to’, you have the option to apply the settings to specific tubes and not to all of them.
Settings > Set Label. Type a label for each channel you use (optional). Click ‘Close’. This will apply to every tube you are going to create or have already created. If you click ‘Apply to’, you have the option to apply the settings to specific tubes and not to all of them.
To create plots, use the following icon:
You can generate histograms, dot plots, pseudocolour plots and contour plots.
To change the parameter on the axis, click on the axis you want to change and find the parameter you want
Go to workspace area and create the plots you want. To create gates, use the control buttons below:
The gates are mainly useful to ensure you will record enough of the cells of interest.
To continue with these cells in the gating strategy, click on the heading of the next plot and select the gate you just created
Now repeat the previous steps to create gates until you have your entire gating strategy
Click on population hierarchy (icon below):
The Population Hierarchy function allows you to view how gates rank in relation to one another. To change the display colour, double-click the default colour and select the desired colour from the drop-down colour palette. To change the name of each gate, double-click the name of the desired gate.
Check if your gates have the right hierarchy especially if you need to record a specific number of a rare cell type.
2. New Experiment from Template:
Select New Experiment from Template
Select Browse next to New Experiment and specify the file path for the new experiment, then select Browse next to Template and specify the file path to the existing template OR
Select the Template tab on the Start page and select the template from the list of recently used templates. Specify the file path and save the experiment.
3. Open an existing experiment:
Select Open Experiment, specify the file path and save the experiment.
Select the Experiment tab on the Start page and select the experiment from the list of recently opened experiments. Specify the file path and save the experiment.
Running a sample
Select the icon below from the Test Tube screen to create the new sample tube. The first sample tube is already created by default.
To change the name, right-click on the tube or simply double-click on it and select ‘Edit Name’.
Vortex the sample (FACS tube or Eppendorf)
Ensure that the sample tube holder is in the sample loading position. If not, select Initialize. Place the sample tube in the sample tube holder.
Select the desired acquisition parameters (Events/Time to Record and Sample Flow Rate) on the left side of the screen. Three stop count collection conditions are available for sample recording:
Events to Record. Used to set the number of events to record in the specified population.
Time to Record. Used to set the collection time duration in second.
Volume to record. Used to set the collection volume of the sample.
Select ‘Run’ to load the sample
View the plots and establish the gates.
To adjust axis ranges, use the axis display controls located at the top of the screen.
Select the following icon to zoom-in and define which area of a plot to enlarge. The selected area can be magnified to fill the entire graph. By selecting the zoom-out function, you can click on the graph and restore the plot to its original appearance before magnification.
Select the icon below to shift the axes. The mouse pointer appears as a hand. It allows you to drag the graph to reveal the axis segment you need.
NOTE:
‘Pan’ icon modifies the axis display range dimensions when panning both axes. When the pan control is selected, you can right-click the graph and select which axis you need to adjust when dragging. You can also pan directly to the default axis range.
‘Single side pan icon’ modifies the axis display range dimensions when panning one axis. NOTE: Only the low end of the axis can be adjusted by the single side pan tool.
Select Record to save the data.
Saving the experiment
Go to ‘File’ tab > Save to save the experiment
Go to ‘File’ tab > Save as to save the experiment under a different name as a backup
Go to ‘File’ tab > Save as Template to save the template
Exporting FCS files
Go to ‘File’ tab > Export FCS files. A window will appear having the list of the tubes you run.
Select the tubes to export and click ‘OK’
Select the path to save the FCS files.
Select ‘Next’
Setting up an experiment (plates)
Set up the experiment in CytExpert software
Open CytExpert software (CytExpert desktop icon).
The Start-up screen appears.
To turn on the cytometer go to ‘Cytometer’ tab and select ‘Turn on’
Verify that the connection indicator light in the lower left corner of the software screen is green, and Connected is displayed. The left side shows the connection status, the middle shows the instrument status, and the right side shows the status details. If you see red indicators, see ‘Troubleshooting’.
Verify that the Sheath and Waste flow indicators in the lower right corner of the software screen are green indicating that the fluidics system is normal.
Selecting the Proper Sample Injection Mode
To run sample in plates, follow these steps:
Go to Cytometer > Sample Injection mode > Plate Loader.
The following message will appear. Press ’OK’ and turn off the cytometer (Cytometer > Turn off). Then, turn the cytometer back on (Cytometer > Turn on).
Check the metal twister above the sample holder is on plate mode (see picture). If it is not, twist it the way it shows ‘P’.
Running the System Startup Program
If you are the first person using the instrument in the morning, you should perform ‘System Startup program’.
Go to Cytometer > System Start up Program
The plate loader automatically ejects the plate holder stage and the System Startup Program window appears.
Follow the on screen software prompts and select the desired wells and select Set As Deionized Water Well.
Prepare a clean wash plate.
Select Load to load the plate.
Select Start to start the program. Select OK to The message ‘Please confirm that the correct plate is placed properly’.
When cleaning is complete the plate loader ejects the plate holder stage. Select ‘Close’ to quit the startup program.
Creating an experiment
Refer to ‘Creating an Experiment’ in Chapter ‘Cytoflex LX – Setting up an experiment (tubes)’.
Select the following icon
The Plate window appears:
Select the Add Plate dropdown
Select Add Plate.
Select the Plate type from the dropdown
Select the direction of the workflow from the sampling sequence from the plate section of the window.
Select OK.
Setting Sample Wells
Once the plate protocol is created, the plate window appears.
Left click and drag your mouse to highlight the desired wells or hold the Control key and select each desired well.
Select the following icon
or right-click the selected wells and select Set As Sample Wells.
The Set As Sample Wells window appears
Enter the name in the Prefix box in the Naming Rules section of the window.
Enter the Group Name in the Name box in the Group section of the window.
Select the sample well colour using the colour dropdown under the Group section of the window.
Select the channels and create label names under the Channel tab.
Select Events to record or time to record under the Stopping Rules tab.
Select OK
Running samples in plates
To run a single well:
Select the well in the Plate window.
Select Run to prompt the system to begin sample aspiration.
Select Record to save the data.
Select Eject to prompt the plate loader to eject.
To run a set of wells:
Select the desired wells.
Select the following icon or right click and select ‘Auto Record’ to set the selected wells for auto record. Number labels appear in the bottom right corner of each well set for auto record. Sample acquisition occurs in the order indicated by the numbers.
NOTE:
Skip the current well and move to the next well for acquisition.
Pause acquisition. The software completes the current sample well acquisition and pauses when the current well is complete. The plate can be loaded and unloaded while the sample acquisition is paused.
Continue the acquisition.
Saving the experiment
Go to ‘File’ tab > Save to save the experiment
Go to ‘File’ tab > Save as to save the experiment under a different name as a backup
Go to ‘File’ tab > Save as Template to save the template
Exporting FCS files
Go to ‘File’ tab > Export FCS files. Select the tubes to export and click ‘OK’
Select the path to save the FCS files.
Select ‘Next’
Daily cleaning procedure
Daily Clean should be performed every time after using the instrument to clean the sample line.
To perform Daily Clean with tubes:
Cytometer > Daily Clean
Follow the prompts appearing on the screen.
Insert a tube with 2 mL of FlowClean solution into the sample holder and select ‘Run’.
Remove the Flow Clean tube. Select Duration of 3 mins.
Insert a tube with 2 mL of DI water into the sample holder and select ‘Run’ to perform the second step of the cleaning process. Select Duration of 3 mins.
After the process has been completed, remove the sample tube and close the Daily Clean window.
To perform Daily Clean with plate loader:
Cytometer > Daily Clean. The plateloader automatically ejects the plate holder stage.
Follow the on screen software prompts and select the desired wells for cleaning agent and deionized water.
Select Load to load the plate.
Select Start to start the cleaning procedure. The message Please confirm that the correct plate is placed properly and press OK appears. Select OK.
Select Close.
Shut down or Standby
Check the calpendo calendar and choose either of the two options below depending on the calendar booking:
Someone is coming within 3 hours after your booking finishes and before 5pm
Clean the cytometer as described in ‘Daily cleaning procedure’ section
If necessary, empty all waste liquid from the waste container and refill the sheath fluid tank.
Exit the software. The instrument automatically enters the standby state.
2. No one is coming after you within 3 hours or the next user is coming after 5pm
Clean the cytometer as described in ‘Daily cleaning procedure’ section
Leave the instrument on ‘Standby’ and ‘Low’.
Troubleshooting
Cytometer and software disconnected
If the connection indicator light in the lower left corner of the software screen is red and displays Disconnected and Error:
Make sure you have turned on the cytometer. If you have already turned on the cytometer go to the next step.
Switch off the system from the plug, wait for 10-20 secs and switch on again.
Alarm is beeping
Check the indicators for waste and sheath tank
You can mute the alarm by clicking on Mute Alarm
If the indicator for waste tank is red, you need to empty it. (See ‘Start the Cytoflex LX’ Chapter)
If the indicator for sheath fluid is red, you need to top up with ddH2O. (See ‘Start the Cytoflex LX’ Chapter)
The sample tube holder cannot move up and down automatically
Ensure that the sample injection mode in the software is in Semi-Automatic Injection mode (See ‘Setting up an experiment (tubes)’ Chapter.
The sample flow rate is unstable
That might mean that the sample probe is clogged, maybe because of the fact that the sample contains aggregates or clumps. Or it may indicate that there are air bubbles in the flow cell. To fix the problem:
Run Prime. Select Prime from the Cytometer Menu to prime the flow cell. Wait for the beep and for the Instructions window to close. Otherwise, look for the status bar to display that priming has completed.
Run Daily Clean. (See ‘Daily cleaning procedure’ Chapter)
Filter the sample using an appropriately sized mesh aperture filter.
Populations are drifting
That might mean that there are air bubbles in the system. To fix the problem:
Run Prime
Ensure that the sheath fluid harness and/ or waste harness is not kinked.
No data acquisition
If you cannot see any events:
Make sure the threshold is not too high
Make sure the gain is not too high
Check that the sheath fluid harness and/ or waste harness is not kinked
Run Prime
Reinitialize
Verify that your sample does not have excessive debris. If it does:
a. Filter the sample using an appropriately sized mesh aperture filter.
b. Restain the sample
The sample is flowing, but no signal appears in the plot
Check whether the signal is outside of the display range. Use pan or single side pan icon to modify the display range
Make sure the parent gate is positioned properly and does contain events.
Check whether the threshold is too high and decrease it if needed.