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Pre-Sort Considerations

Before Sorting

Protein Concentration

     Most cells require protein in their culture media to thrive, and the preferred concentration of this can vary greatly.  For samples that will pass through the cytometer however we need to be mindful that the protein concentration in the media used is minimal.  The sample will be introduced to the flow cell via a process called Hydrodynamic Focusing, where an outer sheath fluid will carry and focus the core stream of fluid that is bearing our cells.  The Core stream is comprised of whatever media you prepare your cells with and the cells themselves.  The sheath fluid is usually a saline type buffer, isotonic and protein free.  Each distict fluid will have slightly different refractive indices, that is to say, photons will pass through these fluids differently.  If these differences are great we can end up with an issue known as the Schlieren Effect, whereas more photons will be scattered at the interface of these two different fluids and this can have a deletrious effect on the measurements we are striving for.  In light of this we recommend you prepare your sample media with no more than 5% protein(BSA, FBS, etc) and if possible use less.  The lower the concentration you can get away with the better measurement we'll be able to take.  Having said this, the Schlieren effect is usually not a debilitating factor for most samples, it is most important for measurement of very small particles.  If your particular cells absolutely need a greater concentration of protein to insure viability then by all means bring your sample with the required amount of protein, we have seen samples where the cells rapidly died with lower than 10% protein but this is rare.

Phenol Red

     Your culture media may contain the pH indicator dye Phenol Red.  The color of your media will change from a red color at a pH around 7.4 to a pink color when more alkali and a yellow color when more acidic.  Its is important to note that this dye has flourescent characteristics and will absorb and emit photons spectrally overlapping some of the fluorochromes we commonly use, thus interferring with the measurements we will be taking.  Additionally the refractive index of Phenol Red changes in relation to pH, further adding to complications in regards to the Schlieren Effect mentioned above.  In light of this it is recomended that you do not bring your sort sample in media containing Phenol Red.

Buffering

     Most culture media is buffered using bicarbonate contained in the buffer and CO2 that is pumped into the cell cuture incubator.  This system works well for cell cuture but there is no CO2 being pumped into our sorter so if you rely on this mechanism your sample will be unbuffered.  Many cell types are very sensitive to pH changes and poor viability can result in situations where cells are not properly buffered.  It is recommended that your basal media be buffered using the appropriate concentration of HEPES buffer for sort samples.

Temperature

     Please bring your prepared sort samples on ice.  The sample chamber on the sorter is maintained at 4 degrees Celsius as is the collection apparatus for the cells post sort.

Viability Dyes

     Whenever possible it is recommended that your add the appropriate dye to measure cell viability to your sort sample.  In general a concentration of 1ug/ml of Propidium Iodide, 7-AAD, or DAPI will be sufficient to discriminate dead and dying cells from the viable cells.  Light scatter alone is a poor method to discriminate live/dead, you will get most of the dead cells removed from your gating hierarchy but you will have some remaining always.  The issue regarding these dead cells is that with their unintact membrane most antibodies and fluorochromes will stick to them, creating false positives that you will have difficulty excluding from your target population without a viability dye.

Filtration

     All sort samples must be filtered through 40um mesh filters immediately prior to sorting.  If you are concerned about cell loss due to filtering we can assist you in methods to maximize recovery but I assure you, anything that will not pass through the filter will most likely clog the instrument.  This will result in a downtime that will increase the stress on your cells, and our instruments and staff.

Cell Density

     For lower pressure, 100um sorts you can bring your cells at a density of 5x106 cells/ml to start.  For higher pressure, 70um sorts you can bring your cells at 1-2x107 cells/ml.  Please bring extra sample media in case we need to dilute your samples.  We'll make suggestions as we progress with your sorting to optimize cell concentration to maximize efficiency and minimize sorting time.

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This page was last updated September 26, 2007 and is maintained by Kevin Weller