Flow cytometry

Maintenance

The most important part of maintenance is to keep the cytometer clean.
- Use the flow cell cleaning solution 1X/day when doing experiments.
- Run the "sanitize SIP" protocol between runs.
- Use 10% bleach as cleaning solution. The final concentration of bleach should be 0.5% sodium hypochlorite.
- Inspect the lines to make sure they're not cloudy every time.
- For bacterial samples, run an extra cleaning protocol with 10% bleach or cleaning solution before and after finishing the experiment, and after running the performance tracking beads.
- The "rinse" protocol just cleans the inside of the probe.
- You can and should also wash the probe in the autosampler.
- Run deep cleans and unclogs between users and experiments.
- At the end of the day - 3X deep clean + shutdown protocols.
The autosampler, flow cytometer, and computers should be powered off once a week.
The focus fluid filters should be replaced every three months.
The sample syringe in the flow cytometer should be replaced every 6 months, and keep an eye on it to make sure it's not salty, cracked, or having jerky motions.
The syringe in the autosampler should be replaced once a year.
Compartments should be wiped dry after changing out fluids and waste.
Log in and out of the software every day.
Data should be backed up immediately.
Fastest support: 1-800-955-6288 help line at Thermo.

If the instrument is off, then switch the autosampler on first, then the cytometer, then the computer, then open the software.
When it's full, there is enough sheath fluid in the compartment to do 6-8 hrs of continuous flow.
To replace fluids and remove waste, disconnect the black cable first (sensor) and the fluid cable second. Re-attach in the opposite order.
Inspect the lines to check that none are visibly contaminated.
Log into the software.
Go to the instrument tab --> Startup button. It'll take 3-4 minutes to start up, and once a month it calibrates the autosampler and takes a few minutes longer.

Performance tracking beads are a mixture of small and large stained and unstained beads that are in a small dropper bottle in the 4C fridge.
The performance test needs to be run once per day that flow experiments are being run.
Check that the beads lot on the bottle matches the lot shown in the software.
Check the instrument configuration in the software to make sure it matches reality.
Vortex the beads and add 3 drops of beads to 2 ml focusing fluid in a tube, put the tube in the tube holder, and run the test.
Check the delta PMTV numbers and check for a robust %CV (should be less than 6-7%).
If the delta PMTVs are high, run the debubble protocol.

Use single color controls.
Go to New Experiment --> Tubes. You can also set up compensation in plates depending on what is convenient for your experiment.
Under "Instrument settings", uncheck the boxes for lasers that you're not using and fill in the labels.
Usually, height for bacteria and area for larger cells works better, but you can run both and compare.
Background fluorescence: negative gating can be used with any type of compensation sample, and unstained control is when everything is the same source (cells OR beads).

In the collection panel, set the acquisition volume and flow rate, and then adjust voltages. Start with low settings for volume and flow rate. Make sure the dead volume is accounted for in the total sample volume.
Adjust voltages and axes to place the negative population above 10^2.
Collect data, record, and adjust gates.
Go to Compensation tab --> View matrix.
In the Workspace tab, you can add an all events density plot to look at the cells in FFS vs SSC.
Add a gate and additional dot plot to see the gate FSC-A vs FSC-H. You can run unstained cells and adjust the voltages to see all the cells and fix the gate, then set the gate for singlets, and then set a threshold so that the gated cells are ~70% of the events.
The "Edit gates" menu allows you to change more than 1 gate name and color etc. at the same time
If necessary, Options --> display stain and name for graphs and axes.
Dot plots allow backgating.
Statistics --> hierarchy to see a table of how many cells fall in each gate.

New experiment, with plate setting on the correct type of plate.
Use the plate map to assign samples.
You can set experiment settings with a manual well, and can also add tube samples to groups in a plate
For compensation, you can tell the plate map which wells are for compensation, and these will also be manual wells
If there is a bubble, acquisition will be stopped.

We can use round-bottom plates, V-plates, and flat-bottom plates, in 96- and 384-well formats.
We can also use tubes, including regular 1.5 and 2 ml Eppendorf tubes.

Back up data ASAP. The files are huge. We will aim to do computer maintenance monthly to defragment and delete data.
Options --> customize software allows for user management, like adding users.
Admins can access anyone's data but have to change the password to do so.
Templates for experiments with all the custom settings can also be saved and used on starting up a new experiment.