Flow Cytometer Setup: Instrument Considerations
Francis L. Battye
The Walter & Eliza Hall Institute of Medical research
To a greater extent than is the case with other lab instrumentation, the correct setup of a flow cytometer and the consequent quality of the acquired data depends on the skill and competence of the operator. A mandatory rule is that control samples must be run prior to every experiment. Important general, instrument-related issues that are not specific for any particular application, include the following:
- Laser Alignment: In those cell sorters where laser alignment is not fixed, accurate alignment requires a sample of very uniform test particles. Optics in the vicinity of the saline stream need to be clean (particularly of salt splatter).
- Time Delay: The time delay between signals excited by non-collinear lasers is affected by sheath flow (and therefore by sheath pressure, impedance through sheath filters, etc.) and may therefore be variable and must be checked.
- Sensitivity setting: When acquiring wide dynamic range signals like immunofluorescence on conventional analog-electronics cytometers, it is usual to choose a fixed logarithmic scale. Then the decision is made to devote a fixed region of the scale, e.g. the 1st decade, to the extent of system noise. This provides a limit, above which signals can be ascribed to the fluorescence of applied fluorochrome. The “background” area is also targeted when setting fluorescence compensation. On digital systems with no fixed log scale, a more elaborate procedure must be used. Ensuring repeatability of light scatter and fluorescence intensity results, as demonstrated for appropriate control samples, is a goal of effective instrument quality control.
- Fluorescence Compensation: The choice of valid singly-stained controls is important but sometimes tricky. Directly fluorescinated beads are good controls but only for a limited collection of fluorochromes. Possible subtle effects of he limitation in conventional instruments to pairwise compensation settings should be recognised as should the increased background that arises from high spillover levels.
- Gating: Gating to exclude what are thought to be invalid events (e.g. due to doublets or clumps of cells, to debris or to dead cells) should be applied with caution. Appropriate doublet exclusion procedures are instrument-dependent, as are, to some extent, dead cell techniques.