Very few laboratory instruments combine complex fluidics, optics, and electronics to the extent that flow cytometers do. As a result, these are expensive and challenging instruments to maintain, but sticking with these best practices to flow cytometer cleaning and maintenance can help not only preserve the life of your instrument but also ensure the quality of daily sample analysis.

Cleaning, Cleaning, Cleaning

The sad truth is that cleaning the fluidics pathway of a flow cytometer or cell sorter is a thankless task but is critical for instrument maintenance and performance. Every vendor has its recommendations for cleaning protocols, but here are some key points to consider when developing your standard cleaning protocols.

1. Daily cleaning before every experiment is critical and simple to perform- 5 minutes of FACS clean, 5 minutes of FACS rinse, and finally 5 minutes of DI water.
2. Long Cleans should be performed at least once a month or more if the instruments are in heavy use. A typical deep clean protocol looks something like this-10-15 minutes of 1-5% Contrad 70; 15 minutes of 70% ethanol; 30 minutes of DI water and finally sheath fluid for 10 minutes. It is important that all of these cleaning solutions are used in both the sheath tank and flushed through the sample injection port (SIP). It is also good practice to bypass the in-line filters during a deep clean since the cleaning components can compromise the status of the filters over time.
3. Cleaning the SIP with a 10% bleach solution is also commonly used but must be followed by running DI water for 5-10 minutes after this to remove all traces of bleach before use.
4. Shut-down procedures also employ standard cleaning protocols, typically involving 10% bleach, 10% FACSClean, 70%ethanol, and DI water, each for 5 minutes. HTS units should also be cleaned this way.
5. Contrad 70 solutions should be used with care since the caustic potash component can erode the surface of the flow cell and rubber seals- so always make sure exposure time is limited and all Contrad-containing solutions are thoroughly flushed out of the instrument.
6. There are also instances where additional cleaning procedures might be needed, such as after using Hoechst 33342 or propidium iodine stains. If you process many samples stained with these reagents you may find over time that they accumulate within the flow cell, resulting in failures in % Robust CVs, especially in channels of the UV or violet lasers (Mikes, 2013).
7. Cleaning Checklists can be helpful to ensure best cleaning practices are adhered to, especially for heavily used instruments with multiple users.

CS&T

Bead-based performance systems are now the gold standard for daily instrument checks. For Beckton Dickenson flow cytometers, this involves the use of Cytometer Setup & Tracking (CS&T) beads coupled with the CS&T software within FACSDiva (Perfetto 2012). The system consists of polystyrene beads that emit either fluorescence at either dim, medium, or bright intensities in all channels, and provided an automated approach to cytometer performance over time. CS&T provides several key parameters which collectively track different performance criteria.

1. The CS&T algorithm calculates the PMT Voltage for each detector and is used to reach and sustain a stable MFI target over time. If the adjustment required to reach this MFI is greater than 50V, then CS&T will fail.
2. Optical Background Noise (Br). The higher this value, the lower the optical resolution of the instrument.
3. The efficiency of the detector (Qr) represents the number of photoelectrons detected per fluorescent unit. A higher Qr value represents higher resolution.
4. Percentage rCV or the robust coefficient of variation for each detector, ideally ~3%.
5. The instrument laser delay. The physical separation of the lasers within the system means that the CS&T beads will intercept with each laser during different segments of time. The interception of a bead with the first laser is ahead of its interception with the last, however, all data relating to this bead will be transferred at the same time. The time delay between the bead’s interception between the first and last laser is referred to as the laser delay- and this is dependent on sheath pressure.

If CS&T fails, it can mean several different things. A review of the CS&T reportable can then be used to troubleshoot the issue. For example, if all the readouts from a single laser are off, then that laser may need to be replaced. One of the most common reasons for a failure in CS&T is a lack of events- either due to a clog, or a pressure issue in the instrument that is preventing the sample acquisition. The clearing of clogs is addressed below. Pressure issues can be addressed by examining the fluidics for air bubbles, confirming that there is sufficient sheath fluid and the waste tank empty. Another common failure is in the bal seal that seals the SIP to the sample tube. It is also important to confirm that there are no cracks in the sample tube that can prevent sample acquisition into the SIP. Failure through PMTV is typically caused by a dirty flow cell or a laser alignment issue; however, both factors can also result in a failure of %rCV.

It is good practice to track instrument performance over time through Levey Jennings plots, and this also meets the requirements for several regulatory checklists. Trends in instrument performance can also help predict future problems, enabling instrument downtime to be minimized.

Clearing a Clog

In the world of flow cytometry “clogs happen”, even when stringent cleaning protocols are employed, and samples filtered. There are some tried and trusted approaches to tackling clogs.

1. Running 3 or more rounds of instrument priming. The priming process forces air back through the lines and into the sheath fluid tube, and the bubbles created by this can help dislodge clogs from the tubing and the surface of the flow cell.
2. Cleaning the nozzle. Since this is the final exit point on cell sorters, larger clogs are frequently captured within the nozzle. We always recommend having a spare nozzle on hand, but if not, then the nozzle can be removed and cleaned with 10% bleach and even sonicated to help dislodge unwelcome cell debris.
3. Turning up the heat! Many technicians swear by this trick of heating FACS clean in the microwave before using it to more effectively flush out clogs.
4. An alkaline daily cleanse can help to remove nucleic acids and cellular debris from instruments. Typically, a filtered solution of 0.1N sodium hydroxide is used, followed by FACSClean or equivalent and a final rinse with DI water.
5. Prevention is always better than cure! Using cell strainers to remove clumps before they end up in your instrument is a no-brainer. For really sticky samples, we recommend including DNase as buffers to remove the DNA from damaged cells.

Final Thoughts

There is now a diverse array of flow analyzers and cell sorters on the market, and no collective routine for maintenance and cleaning of these instruments. Instrument maintenance starts with cleaning- not only the fluidics system, but also the removal of dust from the optical filters, mirrors, and prisms with either compressed air or dust-free wipes. Filters and tubing should be replaced as recommended by the manufacturer or following any incident during which these could be compromised. Most manufacturers provide service contracts; however, an appropriate cadence of maintenance and cleaning is still necessary to help prevent performance issues. Since flow cytometers do not generate quantitative read-outs, the tracking of instrument performance (e.g. with CS&T beads) is important for identifying trends in the output of these complex instruments and troubleshooting a range of optical, fluidics-related, and electronic issues in real-time.

Flow cytometers and cell sorters require a significant investment in maintenance and cleaning. Some of these processes can seem arduous but in the long run, they serve not only to ensure the quality of the sample analysis but also help to maximize the life of your instrument.

References

• Mikes, J. et. al. (2013) Procedure for improved cleaning of FACSAria cuvette flow cell. Cytometry Part A. Vol. 83A, (6) pp. 523-527
• Perfetto SP et al. (2012) Quality assurance for polychromatic flow cytometry using a suite of calibration beads. Nature Protocols 1, 1522-1530.