Expert Cytometry
co-founder
Expert Cytometry
co-founder
Cell sorting can be a scary proposition.
A precious sample is introduced into a machine that pressurizes the cells to 70 PSI, moves them past one or more lasers, vibrates the stream at 90 kHz before decelerating the cells to atmospheric pressure before they hit an aqueous surface.
Many cells survive this journey. But some do not.
Here are 10 things smart scientists do to improve their cell recovery.
1. They pre-coat their catch tubes.
A smart way to improve your cell recovery is to incubate your plastic tubes with a buffer solution containing protein. This will help reduce/eliminate the charge on the plastic.
Since the droplet containing the cell is charged, it can be attracted to the charge on the plastic. This results in the droplet hitting the side of the tube wall, and the cell dying as the small volume of liquid evaporates.
To prevent this, pre-coat the tube with protein/buffer to neutralize the plastic charge. Even better, make sure that your tubes are not polystyrene.
2. They know the catch buffer.
Cells are going to be traveling in a buffered saline. This is not very conducive for keeping cells alive for long periods of time. The good news is that you can improve your recovering by ensuring that the catch buffer has some – but not too much – protein in it. Typically only 10-50% protein in the catch buffer is sufficient.
3. They add HEPES.
If you’re sorting into media, make sure the media is HEPES buffered. Buffers like RPMI are formulated to buffer in a CO2 atmosphere (like the atmosphere found in your lab’s incubator) and, as such, don’t buffer well in our normal atmosphere.
4. They keep them cold (or warm).
Smart scientists know how their cells respond to temperature differences. Some cells do not like to be kept cold and will die quickly if sorted into 4 degree Celsius buffer.
5. They use a soybean trypsin inhibitor.
Sorting adherent cells adds a level of complexity to an experiment. The cells have to be disassociated to pass through the sorter, and this is often done with trypsin. The quickest and most common neutralization method is to add FBS to the cells.
Be careful of this – while it neutralizes the trypsin effectively, it also adds back all the components that cells need to re-adhere to each other. Try soybean trypsin inhibitor instead.
6. They filter.
Nothing is worse than a clogged nozzle when sorting. It adds time to the sort and reduces efficiency (and annoys the sort operator).
Just before sorting, make sure to pass the cells through an appropriate sized filter to remove clumps and debris. The smartest scientists go as far as looking at their cells under a microscope to ensure that there are no clumps prior to sorting.
7. They use a viability dye.
Make sure to include a viability dye in your staining panel. This will help eliminate dead cells.
Using a viability dye is always a smart decision.
8. They design proper antibody panels.
When trying to define a cell population, make sure you include both positive and negative markers in your antibody panel. The use of dump channels, negative markers and multiple positive markers will help ensure that the sorted cells are what they are supposed to be.
9. They ALWAYS count their cells.
Know the cell count at the time the cells are going onto the sorter – NOT from when you first began preparing them.
Since an optimal sort speed is typically ¼ the droplet generation frequency, over concentrating the cells will reduce purity at the back end. Bring some dilution buffer with you just in case the cells are too concentrated.
10. They are aware of their threshold settings.
The higher the threshold, the easier it is to visualize the specific cell population. But this doesn’t eliminate the fact that the debris and junk are still present within the cell population you’re visualizing. It means that the cytometer is ignoring it.
Here’s the key: whatever the cytometer ignores will end up in the final sorted population.
For example, I’ve seen people send a “pure” population of sorted mature B cells in for genomic sequencing only to learn that hemoglobin (from contaminating red blood cells) is the most abundantly expressed gene in their sample.
This article originally appeared on expertcytometry.com. Read more about cell sorting from Tim Bushnell and Carol Oxford here.
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