Being “Accuri-te” Through Cytometry: A Guide to Accuri C6 Software

A new lab toy to make it big in the last 5–10 years is the Accuri C6 cytometer (now under the BD umbrella), a low-cost instrument in comparison to the big boys. Lightweight, with a small footprint and straightforward maintenance, it’s often the cytometer of choice. It may be suitable for those labs that require more routine applications, and/or don’t require a large number of fluorescence channels (488nm [blue] and 640nm [red] lasers, 4 colour detectors). You can change the detector setup, or add on a plate loader, but that’s about the height of the upgrade options.

Accuri C6 Software

Imaginatively, the associated software is called Accuri C6 software. The software is split into four tabs; Collect, Analyze, Statistics, and Batch Analysis. A copy of the program is supplied with the cytometer, and the license allows that it can be loaded on to users’ PCs/ laptops (PC only). This is convenient when you want to do your analysis away from the cytometer. Unlike other programs, it doesn’t require a serial number/ dongle, but it’s only compatible with C6 data. So, it’s no good for FCS files from other instruments alas.

Screenshot of the Collect window from the Accuri C6 software

Acquisition

Collect is the acquisition window, for running samples on the cytometer. It is clear and intuitive with a Control Panel for naming samples (always in 96 well plate format, but don’t let that confuse you!), setting stopping gates, sample fluidics rates, etc. You can create plots (only 3 plot options) on the workspace on this page, and rename parameters as you wish. Data from all 6 parameters (FSC, SSC, 4x fluorescence) are saved with every sample, and with over 16 million digital channels per parameter there is a huge dynamic range which allows for fixed voltages. Not having to worry about setting voltages tends to be a very welcome feature for new users or those prone to AA (Acquisition Anxiety)! Event rates are up to 10,000 per second with a max of 1,000,000 per sample, which is plenty for most applications. If not, you can usually play with the threshold and storage gates to work around it. Regions and gates can be created easily on any plot with associated stats listed on the lower end of the page.

Analysis

The Analyze window lets you create analysis plots (including overlay) to interpret your data one at a time, which is clear and easy to do. You can print the plot, save as image, or drag and drop (as .png or .eps) to other programs such as PowerPoint or PhotoShop, where you can further edit. The Batch Analysis window is probably a little more user-friendly and faster than Analyze. Simply click the plots you want to present/analyse, then the samples you wish to look at (or just select all), at which point they all appear below, and are ready to be exported if you so wish (to PowerPoint or Excel). The plots are maybe not as pretty as through other programs but should suffice for publication. The Statistics window lets you pick and choose which stats, for which populations, you’d like to visualise; and again it is very handy and straightforward. Strangely the median statistics aren’t automatically shown for any stats window- you have to select from the Display dropdown menu.

Saving

Each experiment (with all associated gates, stats, analysis etc.) is saved as one file. You will be prompted to name and assign to folder once you run the first sample. At this point, you should name it something particular to the experiment (e.g. 2015_06_21_CellCycle) rather than specific to the sample (e.g. 10µg_drug_A). If you wish to analyse the data on other software, be sure to export it. And maybe a little confusingly, if you want to analyse it on FlowJo (or some other analysis software), be sure to select “Export ALL Samples to Third Party…”.

Overall

Overall the Accuri C6 software (and cytometer) is very user-friendly and intuitive; though not as sophisticated as others (and not supposed to be). However what it lacks in sophistication, it more than makes up for in ease of use and its price point is amongst the lowest of all cytometers out there. It would probably suffice for most labs that are in the 1 to 3 color world and would like an uncomplicated instrument. For me the Color Compensation window is clunky and harks back to days of having to set it manually. So if using more than one color, I’d suggest either compensating offline post-acquisition or creating a dedicated template for it.

For most users with relatively uncomplicated experiments (cytometry-speaking anyway), this will most likely do the job for you. And be a stress-free, dare I say pleasant, experience- though, we can’t guarantee your results will leave you happy!