Flow Cytometry Lab

Cell sorting creates aerosols that may pose a risk to the operator and others in the sort lab. Cell sorting must be included in your lab’s Biological Safety Protocol and on file with the UW Office of Biological Safety.
Updated 9/18/17

Assay standardization in flow cytometry improves reproducibility and reduces cytometer set-up time. For assays evaluating protein expression levels using MFI (median fluorescence intensity), this standardization is critical for reducing user set-up variation among replicates. For all flow assays, standardization expedites cytometer set-up and data analysis.
Updated 9/18/17

Designing and optimizing a multicolor flow experiment from the ground up takes a significant amount of front-end work to plan and optimize. This development time will save you time and precious sample in the long run!
Updated 9/18/17

It is important to consider the source of aggregation in your samples to properly address the issue. Adhesion can often be counteracted by removing divalent cations. However, the activity of DNAse requires divalent cations. Using EGTA instead of EDTA may allow magnesium ions to interact with DNAse while still partially mitigating adhesion.
Updated 9/18/17

Separation index and concatenation for FlowJo antibody titrations.
Updated 9/18/17

Compensation beads offer a variety of benefits. The most obvious of these is that they allow you to save more of your cells for your experimental conditions rather than controls. Additionally, compensation beads stain more brightly and uniformly than cells, allowing for easy compensation of colors that may be only on rare events in the cells sample. Importantly, compensation beads can be used with the same antibodies you use for your experiment ensuring a perfect fluorochrome match.
Updated 9/18/17

Titration allows you to determine the amount of antibody that gives you the best separation of populations in your samples, and the best measure of expression levels. Too little antibody means the cells expressing the marker of interest do not stain as brightly as they could, and may not separate adequately from the negative cells. Too much antibody can increase non-specific binding, which increases the spread and background of the negative population. Both situations result in lower resolution of the measurement.