Tech Notes

Technical Note describing method and steps for incorporating a bead standard into flow cytometry assays acquired on the Attune flow cytometer.

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Updated 9/18/17

Cell sorting is an aerosol-generating procedure, with greatly increases the risk of airway transmission of hazardous and infectious material. Aerosol incidents are not uncommon during normal sort procedures. These incidents can be caused by sample clogging and other disturbances that increase physical instability of the fragile single-cell stream.
Updated 9/18/17

The cell cycle profile of a sample can be determined by staining the DNA with a fluorescent dye and measuring its intensity. The dye stains DNA stoichiometrically, allowing differentiations of cells in G0/G1, S phase, and G2/M, as well as identification of aneuploid populations. A variety of staining protocols can be adapted for different sample types, but the general analysis remains the same.
Updated 9/18/17

Good sample preparation practices are critical for flow cytometry. Filtering removes large aggregates that may otherwise clog the cytometer, but doublets and small multiplets will pass throug hand remain in the sample. Each clump passes through the lasers and is interpreted as one event with the combined properties of all cells in the group. These composite events must be excluded during analysis.
Updated 9/18/17

Cell viability, autofluorescence and cell aggregation may affect the overall quality of live cell sorting assays. Superior cell preparation is crucial and will result in better sort purity, yield, and post-sort cellular function and viability.
Updated 9/18/17

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