FACS Buffer - (Mar/13/2012 )

Hello all,

I'm a relatively new FACS user, as is everyone in my lab. We've had a basic introduction to flow cytometry and the machine is running relatively well, however, we frequently have problems with clogs. If the nozzle isn't clogged, then there is something blocking (or at least causing problems) with the sample line. We work with adherent cells, and unfortunately everyone has just sort of been using whatever buffer they want when they do FACS. I assume this is part (sample preparation being the obvious other half) of the problem. Could I get some suggestions from experienced FACS users for what a good FACS buffer is and why you add what you add. I have one guy sorting with his regular media, one guy using 10% FCS in his buffer and when I tell them it's not good, they just tell me: It makes my cells happy and I don't have any problems with clogs! Of course, the next person using the FACS more likely than not will have a clog, so I would like to have some feedback from more experienced users. Are they right and anything can be used as a buffer? How do I convince them if they need to use less FBS? I am under the impression that a max of 2%FBS in PBS with 1mM EDTA is the basic FACS buffer. Any help so that we can reduce the amount of time wasted on troubleshotting due to clogs would be helpful!

Sorry, I'm just a lowly tech and it's hard to get some PhDs to listen, ergo I need some solid reasoning behind my argument. Thank you in advance.

Well, in our lab we use 1% BSA in PBS or alternatively 3 % FCS in PBS (personally, I always use BSA). So far, I never had problems with clogging. I know this does not really answer your question, but I thought I'd point out the possibility of using BSA. Did you ever try ?

Do you do a cleaning plate or tube between runs? And have you had a look at the cell suspension they are running through? Perhaps their adherent cell lines are not single cell suspension when they are running?

I'm not saying for definite that the FBS concentration can't be a problem, but it is routinely used in FACs buffers so it can't be that bad. And I've used as high as 5% before with no problems.

The facility where I do my flow is VERY strict about cleaning the machine between runs, so perhaps you could introduce that to your lab?

For what its worth, if you came to me and told me I shouldn't use the buffer I am- but couldn't give me a reason why, I'd ignore you too, sorry

Also, if you leave the instrument not in use for some time, but don't flush out the sheath with water- it can cause crystallisation which can lead to clogging. Our cytometer has a fluidics shutdown (which includes this water replacement) done at the end of EVERY day!

You should certainly have EDTA in your FACS buffer, this helps to prevent aggregation of cells and clogging. I always use 3mM EDTA and 5%FCS in PBS. Another problem are fixed cells (cell cycle), they tend to aggregate anyway. Pipet well and run nozzle cleaning procedure if you have clogging. Which instrument are you using, FACS Canto with Diva 6.0?