little magnetic beads saved my phd
One mainstay method for cell sorting is with flow cytometry. However, this can be a time intensive procedure and depends on the availability of a flow cytometer with sorting capability and someone who can run the machine. And while most major universities have flow cytometry cores with dedicated technicians, who often know waaaayyy too much about flow cytometry, you will have to schedule an appointment along with everyone else in the university who needs cell sorting. At the institution where I did my PhD research, there were several sorting facilities but only one of them could handle my samples and the technician who ran that facility worked from 10am to 3:30pm (what is this, a bank?!?!?) performing only two sorting runs per day. And with the number of people who wanted to use that sorting facility, I was left having to schedule my cell sorting 1 month ahead of time. The problem I ran into was that most of my experiments were dependent on getting purified cell populations from blood samples and when I could get blood samples was quite unpredictable. Plus on top of that, the sorting tech wouldn’t let me schedule more than 2 sorting runs at a time. At the rate I was going, I would be able to do maybe 1 experiment per month and my PhD would take on the order 10 years to finish. Painful. Just painful.
But then one of the older mudphudders to whom I was venting turned me onto cell sorting using magnetic beads and it saved my thesis project. Magnetic beads, attached to antibodies specific for a particular cell surface marker are used to label all cells with that marker and a magnet is then used to pull those cells out of an otherwise heterogeneous population of cells. Brilliant! All of a sudden, my cell sorting needs were no longer dependent on the availability of anyone but myself and I could do my sorting whenever I needed to—day or night. I will point out for anyone interested that sorting with magnetic beads: (1) is probably best characterized for isolation of specific cell populations from blood samples although can be used well with cell suspensions isolated from a tissues; (2) may be toxic to cells, depending on the cell type and the company you buy your beads from (beads from different companies–e.g. Miltenyi or Dynal–are made of different components); (3) will stay on the cell’s surface a lot longer than antibodies used for flow cytometry and may block any receptor/ligand interactions involving the cell surface protein in future experiments with those cells; and (4) usually does not allow sorting for the presence of more than one cell surface marker (i.e. you can only isolate cells that are positive for marker X *and/or* marker Y not cells positive for marker X *and* marker Y), unlike flow cytometric cell sorting—there are exceptions to this, in particular any bead that can be removed from the cells after sorting. Using this technology, I was able to get specific cell populations (sorting on 2 markers, actually) at purities and yields comparable to flow cytometric sorting (after some optimization of course).
When I was starting my PhD, cell sorting with magnetic beads wasn’t nearly as well known as it is today (…definitely aging myself…) so probably everyone reading this is already aware of that technology. but I wanted to mention this story because anytime you are dependent on a core facility for a necessary part of your research but are getting held up by logistics—odds are that there is technology out there that can let you get around it. I think the most frustrating thing is when you want to do work but can’t—and the worst is logistical problems, like when you are working 12 hours per day and you are dealing with a tech who works 5-6 hrs/day (in all fairness, I will say that the techs at another sorting facility that couldn’t handle my sorts were extremely helpful and really taught me everything I know about flow cytometry). You just gotta keep talking to people—graduate students, postdocs and PIs—and looking at product catalogues. If you are getting jammed up by logistics, odds are that many others have too and so some company has probably developed some solution that you need to find.