Many fluorescent protein (FP) variants can be analysed using the Accuri C6. This note provides guidelines for the C6 user performing FP analysis.

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The Accuri C6 Flow Cytometer® simplifies cell analysis by performing absolute cell counts, eliminating the need for counting beads, or dual-platform hemacytometer counts. The microprocessor-controlled peristaltic pump system allows the sample volume pulled per run to be determined. The volume (in μL) appears as data in the CFlow® Software statistics tables, and the counts per μL for any gated population is automatically calculated and can be selectively displayed in the Statistics Tab data view. These guidelines cover the factors to consider when performing absolute cell counting with the C6.

Preventative maintenance

As with any lab instrument, optimal performance requires proper system maintenance. Accuri suggests the following preventative maintenance routine:

• To prevent clogging, run filtered deionized water (di H2O) on fast for three-five minutes at the end of each experiment
• Run decontamination and cleaning cycles each day of use. These cycles are run automatically during machine shut-down
• Keep the C6 wetted by placing a tube of di H2O on the SIP between uses. Note: this is done automatically with the CSampler® by placing the SIP in the wash station when idle or at system shut-down
• Thoroughly clean the flow cell each month by performing an extended flow cell clean (KR-235)
• Replace peristaltic pump tubing (CP-105), in-line sheath filter (CP-138) and bottle filters (CP-132) every two months

Performance validation

Prior to running samples for absolute counting, validate system fluidics performance by running Accuri Volume Validation Beads (QA-120) according to product instructions.

Sample Concentration Range – The C6 will accurately measure volume in samples with concentrations ranging from 1000 – 5 x 106 cells/mL. Concentrations in excess of those recommended may result in inaccurate counting due to system saturation. The appropriate cell concentration range that can be analyzed will vary between cell types, based on considerations such as size, shape and tendency to clump. In addition, the flow rate used, relative to sample concentration, should minimize doublets and larger clumps, and must never allow the event rate to exceed 10,000 events per second. It is therefore recommended that users compare reported concentrations of serial dilutions of their sample to verify a linear correlation.