By Michael Kissner
When setting up a cell sorting experiment, there are many things to consider.
You must consider which controls you’re going to use, which quality control beads are ideal, how you’re going to compensate the experiment, which instrument and which instrument settings are ideal, on and on to how you plan to analyze, gate, and present your data.
With so many things to consider, it’s easy to lose site of the small things that can drastically affect the viability of your cells, including the composition of your suspension buffer.
The composition of the suspension buffer for preparation, staining, analyzing and sorting is perhaps the most important parameter for maintaining viability during a cell sorting experiment.
While the precise components of a buffer can differ depending on the cell type, there are 5 key points to keep in mind…
1. Cell culture medium is usually not a good choice.
Most cell culture media are formulated with a CO2-carbonate buffering system that is optimized for tissue culture carbon dioxide partial pressure, which is higher than the typical ambient carbon dioxide partial pressure.
When these kinds of buffers are left in typical atmospheric conditions, carbon dioxide will evaporate from the medium and cause the pH of the medium to rise into the alkaline range (in phenol red-containing formulations, the color will become more purple), which expectedly can significantly affect viability.
While tissue culture medium is often used as a suspension buffer without deleterious effect, longer cell sorts and the sensitivity of the cell type can certainly exacerbate the effect of pH on viability.
Additionally, although it’s important to formulate the buffer with some kind of protein, tissue culture media may contain serum at concentrations of upwards of 10-15%, which can be deposited over time on the sample line and cause flow rate irregularities during the sort.
If tissue culture medium must be used, a HEPES-based buffering system, which is not dependent on carbon dioxide, is a preferable and safer choice.
2. Consider adding protein to your buffer.
PBS or HBSS can work well as a base for the suspension fluid, but you should add some kind of protein, typically 1-2% BSA or FBS, to keep the cells happy.
Incidentally, HBSS is formulated with a CO2-carboante buffering system, but this system is meant to work at atmospheric/ambient CO2 partial pressure rather than tissue-culture CO2 partial pressure.
3. Add EDTA to prevent clumping.
EDTA in the suspension buffer, at 1-5 mM, can be a panacea for samples that tend to form aggregates and clog the instrument. This molecule interferes with cell-cell adhesion molecules by sequestering the divalent cations that these molecules require to function.
Therefore, it can greatly reduce the incidence of clumping in the sample. Preventing clogs has an indirect effect on viability—clogs cause downtime during the sort, requiring cells to remain in suboptimal conditions for longer than necessary. The faster the cells are manipulated, the lower the likelihood that they will succumb to apoptosis or necrosis.
If you will be using DNAse (see #4 below), then EGTA is a better choice for chelation to prevent clumping. EDTA will chelate Mg++ ions, which are necessary for DNAse, while EGTA has a much lower affinity for Mg++.
4. Add DNAse to deal with dead cells.
Certain kinds of samples, especially those harvested from some kinds of solid tissues, will inevitably contain a high proportion of dead cells. If this is the case, it may be advantageous to add DNAse to the suspension buffer used for the sorting process.
Dead cells will release genomic DNA into the buffer, which tends to aggregate and form clumps. These clumps can trap cells, cause clogs and result in an overall drop in recovery after the sort. Recommended concentrations vary, but 200 µg/mL has been reported in Current Protocols In Cytometry to be effective. Alternatively, debris from dead cells can be removed during the preparation either through centrifugation gradients or depletion via magnetic separation.
5. Keep everything at the right temperature.
The temperature at which the sample is maintained during preparation, staining, and sorting is another important parameter to keep cells alive. The proper temperature strongly depends on the cell type and application.
While many researchers have success preparing and sorting cells at temperatures around 0°C, this may not be the ideal temperature for every cell type.
Nevertheless, there are some general benefits for keeping the cells on ice. Most importantly, a low temperature will slow down metabolic activities and will prevent biochemical or molecular changes that may affect cytometric or downstream results.
For example, some cells, under certain conditions, may shed or internalize antibody-bound cell surface receptors, resulting in diminished staining and resolution on the cytometer. Keeping cells cold can significantly reduce this effect.
Additionally, under the stress of preparation, staining, and the sorting process, some cells may undergo molecular changes—gene expression or otherwise—that could affect downstream experiments or analyses. Keeping cells cold will slow down activity and possibly mitigate any transcriptional effects brought about by the experiment.
Finally, stressed cells may begin to apoptose, lowering your recovery. By keeping your cells cold, you may slow down or mitigate this process so they can be deposited in the collection buffer before progressing further through this pathway.
When designing your flow cytometry experiment, check the literature to see if any particular suspension buffer and temperature conditions have been successful historically in other protocols for the particular cell type you are working. Buffer with protein, EDTA, and DNAse is often a better choice than using your cell culture medium alone. In terms of temperature—cold, usually 0°C, is a good place to start, but temperature can be surprisingly cell-type dependent, so make sure you do your research.
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