Biexponential display

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Compensation tip

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FMO controls

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Annotate your experiment to score points

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Viability marker

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1 potato, 2 potato

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Why wash?

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Centrifuging fluorescent antibodies prior to using them may remove aggregates resulting in cleaner data

Sometimes you may notice a number of events above the obvious positive population, shown below. On occasion these may be fluorescent molecule aggregations that are super bright. 1 trick (and I hope to upload a before and after example when I dig it up) is to either individually centrifuge antibodies before using them, or centrifuging the cocktail before adding to cells in order to remove fluorescent aggregates. In a micro-centrifuge, 10000-12000g, chilled, 5 minutes can be used to pellet aggregates, followed by pipetting the supernatant.

Ensure FSC-A, FSC-H, FSC-W & SSC-A, SSC-H, SSC-W are collected

The digital instruments at WRHFlow allow the collection of additional parameters that can be used to distinguish cell doublets and aggregates of cells from single cells before enumeration.

Avoid clumps in your sample - filter them through a mesh filter

Flow cytometry is a single cell analysis platform (for the most part). Clumping cells can ruin data acquisition as they may clog the narrow orifice the sample needs to travel through to enter the flow cell resulting in incorrect signals being assigned to events due to the way the instruments works. Filtering your samples before running on the cytometer can assist in minimising this occurring. Other things to try might include adding 2mM EDTA into your buffer, enriching cells from debri/dead cells/free DNA before acquisition, keeping your sample cold, and diluting your sample. 

Good practice includes having a time vs. fluorescent parameter (off the last laser used) to monitor your acquisition to spot problems and fix them before finishing running your sample.