Cytometry and Antibody Technology

The Silver Lining of 2021

by | Dec 14, 2021 | Chronicles of an SRL | 0 comments

Let’s not kid ourselves: 2021 was a challenging year for flow cytometry in the CAT Facility. We started out strong early on with most of our instruments available after a very long lockdown. But several sorter issues badly crippled the service, and a flood put half of our benchtop analyzers out of the game for several months. I’m pretty happy to leave all of that behind us!

But this is not the full story — far from it. Through it all, we made some acquisitions that I think will be useful to our users and elevate the quality of the work done in the facility. Believe it or not, 6 new pieces of equipment were obtained by the CAT Facility in 2021!  This was done through the helpful support of the ROCC, the OSRF and, of course, the insurance group that’s helped us getting back on our feet. My thanks to everyone.

In chronological order of acquisition:

1 – GeminiBio Moxi V. The Moxi V is a Coulter Principle-based cell counter that uses a microfluidic cartridge to count and measure the size of cells. It is also equipped with a single photodetector that can be used to assess cell viability using Propidium Iodide. The main reason we got it was to help with the single cell sequencing workflow, and make sure that said cells are in good shape. This is an effort that we’ll pursue going forward. That said, I received enough request from users looking to measure cell size that we thought it would be an asset to the CAT Facility regardless. At this time, we’re not charging for usage on this device, because there’s just no way to track it. We do purchase the cartridges at the lowest cost we can find, and re-sale them with no markup.

2 – BioLegend Mini ELISA Plate Reader. Once upon a time at the UoC, the Flow Core Facility merged with the Fitch Monoclonal Facility and we got two ELISA plate readers. We hardly ever do any ELISAs, but they came in handy every now and then. Suddenly, and for no apparent reason, both of these units died in 2021. We replaced them with the BioLegend Mini ELISA Plate reader and found that it was a neat, cost-effective, easy to use device. I think there are very few plate readers available on campus, so let us know if you need to borrow ours.

3 – ThermoFisher Bigfoot Cell Sorter. We’re looking to replace our aging cell sorters. There are few options available on the market if you’re trying to move away form the BD experience, so we got very excited when the Bigfoot was released in 2020. Our system was installed in September and has been in use ever since. There’s a number of great features available. The plate sorting is just amazing. The flexibility in the use of collection vessels is great – up to 6 populations, or four 15ml conical, or a mix of three 5ml tubes and a 50ml conical, etc. And the tech support has been very responsive to our requests. At this time though, I’ll say that the platform is still under development. Some major features that we will need as soon as possible are not working completely well yet. That would include the 70um tip, which the system has problems handling. We’re also puzzled by the efficiency we observe during the sorts. There’s a few things that could be said here – it may be that we don’t have enough experience yet with the platform, or that we don’t have a proper way of comparing the efficiency between the platforms altogether. So what should we say? I’m confident that it will become a great cell sorter in due time, but it will require some patience from us early adopters.

4 – 5-Laser Cytek Aurora. We replaced our 5-laser Aurora destroyed in the flood for another 5-laser Aurora. Woo. The upshot is that it allowed us to upgrade the plate loader to something that may get better usage. The new plate loader allows samples to be picked up from any type of plates, but also from a 40-tubes plate rack. It vortexes and washes between each sample. Overall, it just feels way more reliable than the previous version and I imagine that many users will find it trustworthy enough to process samples while they do something more interesting than feeding tubes to a machine.

5 – Agilent Penteon. We did not replace the BD Fortessa X20 with another BD Fortessa X20. We decided to go with what we think is a much better platform: the Agilent Penteon. We’ve had it running for a few weeks only, and I’m already so happy it’s here. The user interface is intuitive. The NovoSampler Q is just as great as the equivalent sample loader on the Aurora. And because the Penteon uses SiPMs (remember what Bert said), it is quite improbable that you’ll saturate the detectors and thus you can easily acquire great data without having to fiddle with the gains set by the manufacturer. On top of that, the maintenance of the instrument is automated and there is very little chance that users will end up damaging the instrument by running the sheath tank dry – thus damaging your own chances of getting quality data.

6 – BD AriaIII. We lost the AriaIIIu as well in 2020. What happened there was a cascade of somewhat manageable problems that compounded in a giant mess. It started simply enough with the 561nm laser dying out, then there was a fluidic problem, then the computer crashed and we needed to rebuild one – that proved to be pretty difficult due to the fact that we had to use a Windows XP platform to maintain connection with the AriaIIIu. And then parts were taken form the AriaIIIu to maintain the other two sorters in activity. In parallel, the Surgery Department graciously offered us to take their unused AriaIII that had been used for clinical sorting many years before. We decided that the smarter approach would be to bet on the pristine AriaIII and use the parts of the AriaIIIu to restore it. It has been a bit of a challenge so far, mainly with the computer which has been upgraded to support Windows 10 but keeps crashing. When all is said and done, this sorter will have three lasers (405nm, 488nm, and 640nm) and 14 fluorescent detectors.

Special mentions: One special mention should be given to the MACSQuant Tyto. Miltenyi overhauled the acquisition software which makes it feel like a brand new instrument! Gone is the tedious two-positive-markers-on-adjacent-lasers panel requirement, gone is the Arrival Window tabulation. The complexity of the software as been reduced so much that I would say that the promise of a user friendly sterile cell sorter has finally been delivered.

And lastly, did you notice that our website has improved by leaps and bounds? This would be the work of Associate Scientific Director Laura Johnston. The information should be easier to find now – we think you should be able to find in in two or three clicks at most. There’s already a lot of useful information (check out the Resources Library, Boilerplate Texts, Benchtop Analyzers, and Cell Sorters pages), with more updates coming soon. You’ll definitely hear more about the instruments listed above in the upcoming months.

With that, and on behalf of the CAT Facility Staff, I wish you all very Happy Holidays!

A sad end for the AriaIIIu.

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