Ricerc@Sapienza

The BD Accuri C6 Plus is a compact but also highly sensitive flow cytometer. It is equipped with a blue and red laser, two light scatter detectors (forward and side scatter), and four fluorescence detectors (533/30, 585/40, 670/LP, 675/25) with optical filters optimised for the detection of all popular fluorochromes, newer polymer dyes and many variants of fluorescent proteins. Differently from other models, the laser and optical alignments are pre-optimised and locked down and there is no need to adjust detector voltage. All data is collected over a wide dynamic range (7.2 decade) and can be accessed at all time. Analyses relative to a certain sample type and application are highly reproducible and fluorescence spillover of commonly used dyes is predictable. Gating and compensation settings can be digitally modified before or after acquisition and data can be reanalysed at any time to accommodate new research. This way there is no risk of loosing data because of incorrect settings, many features are automated (see software features below) and users do not need to be experts to perform experiments.

The fluidics system is a unique non-pressurised open system. Unlike many other flow cytometers on the market, the Accuri employs a peristaltic pump and patented pulse dampeners to aspirate samples by suction rather than by positive pressure. Thus the sample, that can be as low as 50 uL, can be collected from many types of small tubes that do not need to be sealed. This open system allows the user to access the sample during aspiration enabling platelet stimulation during acquisition and the continuous and rapid measurement of platelet responses. The time-resolved platelet function assays that I have recently developed exploit this innovative design (Stefanini et al., Blood 2014; Stefanini et al., J Clin Invest 2015).

During sample acquisition, the motion of the pumps is used to determine the sample volume that, in turn, can be used to directly calculate absolute cell counts and measure cell concentration without the need for counting beads (Masters and Harrison, Platelets 2014). Because of its accuracy (comparable to the International Reference Method) and ease this technique is becoming the standard method to count platelets in research laboratories. Exploiting this function I have recently established a fast method to monitor platelet life-span in the circulation of transgenic mice, which is an important assay to evaluate the mechanisms regulating platelet turnover (Stefanini et al., J Clin Invest 2015).

Other important features include that the flow cell diameter (200um) is compatible for particles ranging from 0.5um to 50um. The peristaltic pumps are controlled electronically, thus flow rates and core size can be modified digitally. The flow rate ranges from 10 to 100uL/minute and allows acquiring up to 10,000 events/second. Fluidics are cleaned automatically on instrument shutdown, and laboratory-grade water is used as sheath fluid, reducing operating costs.

The BD Accuri C6 Plus workstation includes a monitor and a computer preinstalled with Windows 7 professional operating system and the BD Accuri C6 Plus software. The software is intuitive so there is no need of specialist training or a dedicated operator. Data is acquired through the "Collect" tab that displays on a single page the sample analysis and gating tools, the cytometer status, the fluidics controls, the run criteria and real-time updates of the acquired events and the statistics. The other tabs contain customisable tools for further data analysis, statistics, and batch analysis. Furthermore, BD Accuri C6 Plus software files can be exported in FCS 3.1 format for data import into flow cytometry analysis programs such as FCS Express software and FlowJo software. To validate that the instrument meets performance specifications the software includes an automated quality control feature. To remove artifacts of fluorescence spillover the software automatically calculates the compensation settings for the most commonly used fluorescent dyes, which can be adjusted manually by the users depending on their specific needs.