Experimental Design Guidelines

Experimental design is a very important aspect of any flow cytometry/cell sorting venture.  There are a few general things to design and/or keep in mind when designing your experiment.  These are important to the success of your experiment and should be discussed with the operator:

Controls
These are, in general, the controls which will be necessary to bring with you when bringing samples for analysis/sorting at the flow core:
1. Sample buffer  This is important for the Accuri C6, as we use this to determine the threshold.
2. Unstained control(s)  You will need to bring an unstained sample of each cell type you intend to have analyzed/sorted.  How do you know if your samples are fluorescent if you don’t have a known, non-fluorescent sample for comparison?  This also helps us to determine any autofluorescence in the cells of your sample.
3. Single stained control (s)  You will need to bring cells or beads which are stained with the same fluorochrome/dye with which your sample is stained.  Notice that this says single-stained controls; hence, if you have a sample that will be stained with GFP, PE, and APC, then you need to bring a GFP-stained control, and PE-stained control, and an APC-stained control.  These are used for compensation.
4. Fluorescence Minus One (FMO) control(s)  These are controls used in multicolor studies which contain all of the fluorocromes of interest except one: hence, fluorescence minus once.  Thus, if you want to run a panel that has FITC, PE, APC, and PerCP, you would, for example, have a control that was stained with just FITC, PE, and APC (this assumes that PerCP is your color of particular interest).  This is useful to see what multicolor-stained cells look like when missing one particular fluorochrome in a cocktail.  The operator may ask you to bring this control; it is not always necessary.

BDAccuri C6It is important to remember when designing experiments for use with the BDAccuri C6 that only one color can be detected per detector at a time.  Thus if you want to use GFP, which is detected by the FL1 detector, you cannot also use AlexaFluor 488, which also uses the FL1 detector.  Please also keep in mind that the Accuri C6 has only 2 lasers and 4 detectors; these detectors have fairly narrow filter restrictions, and so the range of fluorochromes that will work on this machine is also somewhat narrow.  However, there are still many, many fluorochromes that can be used with this machine to yield great results!
For an idea of some fluorochromes that might work with the machine, you can try the BD SpectraViewer.  This is a good starting place; however, it does not have all the possible fluorochromes available, and it does not necessarily employ the correct filter set used by the Accuri C6 in the flow core.  So, while it is a good tool to give you an idea of what you could work with (especially for spectral overlap) it is still very important to check with the operator when designing experiments for flow cytometry.

BDFACSAria IIThe Aria II has more lasers and more detectors than does the Accuri C6, and so the range of fluorochromes that you can choose for this instrument is greatly expanded.  However, this machine is still defined by the filter sets it employs.  These can be viewed on the BDFACSAria II information page.  There is some room to add fluorochromes not listed on this page, but this requires reconfiguring the instrument.  This can be done, so please do inquire about this if you are interested; however, please be aware that it will add extra time to your run.
One thing to remember about sorting is that sort logic is very important.  Sort logic essentially means the sequence of gates that will be employed in order to define the populations that will be sorted.  Ensuring that sort logic is correct is crucial to ensuring that sorts are efficient, accurate, and yield desirable results.  Therefore, it is not only necessary that you be available to review the sort logic prior to your sort, but that you be knowlegable about the biology of your system in order to make informed decisions regarding the sort logic.  Please contact the operator with any questions or concerns regarding sort logic.

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