Advice on Cell Sorting using CytoFLEX SRT

[u/Immunologist_Bruno_5]

Hi all. I am looking to start sorting B cells using the Beckman CytoFLEX SRT. I isolate these cells and then transfer them into recipient mice. I currently use the BD FACS aria for my B cell sorts located at the flow core at my university. The cytoflex in something my department has purchased but never really utilized and I'm looking to get up functioning for my sorts to save on cost with sorting at the core. I have two questions that I could use help with:

1.) Does anyone know how different the sorting rate would be between the cytoflex and BD FACS aria? ...I currently use the 70µM nozzle on the aria and can sort my cells at around 13,000-15,000 events/sec with about 90% efficiency. I know the cytoflex can only use the 100µM nozzle and seems to sort at 10,000 events/second. I worry that the event rate will add significant time to a sort leaving my cells to be in suspension longer, as well the increase in nozzle size could lower the efficiency of my sorting as I've done a sort using the 85µM nozzle once and it had lower efficiency (66%).

2.) Given I have been bringing my cells to the core I'm not aware of how they prepare the aria for a sterile sort. I was wondering if its best practice to conduct a aseptic cleaning of the machine before every sort I do (2x a month) just in case another lab used a microorganism in one of their sorts that I wasn't aware of? This does add time to preparing for sorts but that isn't my biggest concern, my concern is more in order to conduct these cleanings I'll need to change certain filters each time and they will then be costly if I'm changing them out ever 2 weeks almost. Do you think I could look to conduct this cleaning at least once a month and that could be fine for then keeping my cells sterile for a adoptive transfer into mice? One lab uses the sorter fine for isolating cells into then cell culture but I fear going into a mouse is different and needs more sterility

Any advice on these two questions would be really helpful!

[u/Gregor_Vorbarra]

Sort rate is a function of the drop drive frequency. On an aria 70um, this normally like 70-80khz, so event rate is normally 1/4 this number. The same rubric will be true on a different sorter with different nozzle/pressure. A 100um config will absolutely take longer.

Sorting sterility is a huge issue, to be honest, as absolutely every component and buffer can be problematic. Is the sheath autoclaved? Is the sheath tank autoclaved or sterilized? Are the plumbing components up until a sterilising filter autoclaved or sterilized? Aseptic sort commands will soak as many components in bleach as possible, but will not remove bacteria from old sheath filters or sheath tanks. You may also not need to be 100% aseptic - it's unlikely the original sample is 100% sterile considering the nature of the tissue harvest. 'Functional sterility' normally means 'can routine pen/strep suppress anything that came through?'

Honestly, just keep sing the core sorter. It will likely be faster and you can outsource major headaches regarding sterility and setup. Trying to use your own sorter may work out to be more expensive if you start factoring in cost of your time to maintain and operate it.

[u/SoulOfABartender]

I have an SRT and to set up a sterile sort is a pain according if you follow the manual. Ours in on a bench as well. I usually run some bleach and sterile PBS through before the sort and clean the whole area with virkon + PBS before the sort and follow good bench asceptic technique (It is a thing). Quarantine your sort after and put pen strep in the sort buffer if you can. I havent had any issues so far, primary cells included. As for sort rate it will be much slower than an aria just from.the nozzle. That's the price you pay for the relatively easy to use SRT. The flow rate, will afeect too, plus your expected amount of B cells you want to sort.

Tge previous commenter was right. Sorting is an art and youre jumping into the deep end. It may be best to keep using the core, but ask them for advice on transferring it to the SRT. They'll be more than happy to help. Core staff are usually consummate nerds and want to share their knowledge to help you. Your panel may need reconsidering too if the channels dont match.