The ability to individuate multiple properties of millions of cells on a per-cell basis, and even to sort them according to those properties, has made flow cytometry an incredibly powerful tool. Instrument design advances are continually simplifying and streamlining the operator experience, putting the technology within reach of users from a wide variety of disciplines, from neuroscience research to infectious disease diagnostics.
Yet even before strategizing about spectral overlap and where to draw gates, thought and care need to be put into harvesting cells for analysis or sorting. Basic considerations of what it takes to transition the cells from culture medium to FACS buffer should be given at least equal weight with epitope density and secondary antibody isotype.
Get ‘em off, break ‘em up
Medium: “Obviously for flow you need everything in a single-cell suspension,” says Joel Sederstrom, M.S., flow cytometry core director at Baylor College of Medicine in Houston. This isn’t such an issue for cells—like lymphocytes— that grow that way in culture. But for adherent cells or those that are harvested directly from organs, it’s important to use a Ca++- and Mg++-free medium “because the various integrins on the surface of the cell, and the various attachment proteins, need Ca++ and Mg++ to bind to certain other proteins and substrates.” Even if this alone doesn’t cause the cells to release, it helps prevent them from re-attaching.
Trypsin: If the cultured cells still need help, you may need to use protease digestion, but keep in mind that the cell-surface molecules you’re staining for—and even the cells themselves—may be protease-sensitive. “It’s a double-edged sword,” notes Sederstrom, who recommends using EDTA and keeping exposure to enzyme to the minimum necessary to do the job. Growing the cells in a low-adherent culture vessel can also help.
Mechanical disruption: A variety of mechanical disruption techniques are available to make single-cell suspensions from whole tissue, sometimes instead of and sometimes as a supplement to enzymatic digestion. Soft tissues like spleen or liver, for example, can be rubbed between two etched glass slides in a circular motion, or mashed through a nylon mesh using a syringe plunger. This is generally followed by gently pipetting up and down through a glass pipette to release as many cells as possible. Immediately before analysis or sorting, slowly run the cell solution through a needle and syringe a few times. Some researchers have found commercial tissue dissociator systems helpful. For tissues that are more difficult to disaggregate, it may be a good idea to seek out specific advice.
Washes and Pellets: When washing, spin the cells only as hard and as long as necessary. Decant (rather than aspirate) the supernatant, and take care not to leave the pellet dry. Break up and resuspend the pellet prior to adding more fluid.
Sticky situation
DNase: No matter how gentle you are, cell damage may occur, causing cells to spit out sticky DNA, which can cause clumping. Sederstrom points out that it’s probably not a bad idea to include DNase in the medium, unless its use is precluded by the downstream assay.
Plastic: Just as cells may bind tissue-culture vessels, they can be attracted to tubes. Using polypropylene rather than polystyrene minimizes binding, but Sederstrom notes that these softer tubes don’t always fit the cytometers as well when they’re pressurized. Therefore, many researchers use polystyrene tubes that have been pre-coated with protein such as bovine serum albumin (BSA).
In and out: Sederstrom says protein should be in the medium anyway, because it “helps keep everything in a single-cell suspension and also keeps cells from sticking to plastic.” Using undialized serum, on the other hand, will contribute Ca++ and Mg++ to the medium.
Etc: There are other things to be wary of, as well. Biotin can compete with biotinylated antibodies. Phenol red and other molecules, such as flavins, can autofluoresce. Some reactions (like the Annexin V apoptosis assay) can’t be run in Ca++-free media. And azide (found in the buffers of many commercial antibodies) can interfere with sorted cells’ ability to proliferate.
Sort of
Preparing cells for sorting by flow cytometry otherwise parallels preparing them for analysis, with some (mostly) obvious exceptions. Among these are not to fix the cells, which is common for analysis, especially when looking at intracellular proteins; to keep them sterile, which is not necessary for analysis; and to use a medium that keeps the cells happy, which may include high concentrations of serum, notwithstanding the caution above. All of this assumes the goal is to have a pure population of viable cells. But if extraction of RNA or DNA is the goal, (lightly) fix away.