Flow Cytometry: Analysis
Flow cytometer analysers commonly use lasers and arrays of photosensitivity photo-multipliers tubes to record light scatter and fluorescence information from cells in suspension. This technology lends itself, when used in conjunction with specific fluorescent probes and conjugated antibodies, to a vast range of applications. Some of the more common applications include cell cycle measurements, apoptosis studies, monitoring phenotypically distinct populations, RNA expression and cytokine profiling.
The main strengths of cytometers are their speed, with the capacity to analyse thousands of cells per second, and their ability to make measurements at the single cell level, allowing the investigation into the nature of distinct populations which may otherwise be masked by bulk related techniques such as Western Blotting.
The facility manages a suite of analysers (a Canto II, LSR II, LSR-Fortessa X20, Novocyte and Imagestream MKII) strategically sited around the Institute. The LSR II and LSR-Fortessa x-20 are capable of simultaneously detecting forward scatter (size), side scatter (complexity) and up to 18 fluorescence parameters allowing researchers to put together precise, robust panels aiding in the characterisation of rare cell populations.
To increase productivity the Canto II uses a 40 tube carousel system and the LSR-Fortessa X20 a high throughput system (HTS) capable of auto-sampling from 96 and 384 well plates. On the LSR-Fortessa a 96 well plate in standard mode can be completed in 45 minutes or 15 minutes when sampled in the high-throughput mode with little need of supervision.
When robust panels are required to interrogate individual cells across a population by labelling multi-parameters thus being able to ask quite complex questions regarding concentration, interactivity and co-localisation the team offer a panel design consultancy service where appropriate labels and analysis can be provided.
Off-line analysis is supported by Flowjo, a sophisticated software package or IDEAS in the case of Imagestream data.