Spectral Unmixing

Importance of Cell Sorting and Flow Cytometry

Cell sorting using flow cytometry has become indispensable in immunology research. How does this work? A single cell suspension containing heterogenous mixture of cells is focused in a stream of fluid and interrogated by multiple lasers. The emission signals and pre-selected parameters such as cell morphology, physiology and protein expression, separate cells into viable and pure fractions. Thus, sorted cells become a launchpad for numerous downstream studies such as functional assays and cell line development for therapeutic research and development.

Various methods of cell sorting include electrostatic droplet sorting, plate sorting, or magnetic sorting. Typical cell sorters available today have some additional challenges such as affecting cell mortality, long run times, and low purity rates due to lack of precision in plate deposition mechanisms. Ability to sort variety of cells from a heterogenous mix of cell populations with high purity is necessary.

Conventional vs Spectral Analysis

There are two ways to analyze the light emission collected during the sorting process: conventional compensation, or spectral unmixing. The conventional compensation approach applies compensation to account for spectral spillover from one fluorophore into another fluorophore’s detector. This limits the number of parameters which to separate cells. However, immunologists today need to study cell subsets beyond major immune subgroups, effector, and memory cells. Samples in immunology are often limited and complex such as blood, or patient tissues; hence high-parameter experiments to collect maximum data (high content) from smalls amounts of samples in flow cytometry is essential.

Spectral analysis, which uses spectral unmixing to separate various fluorophore signals, fits the bill as it analyzes the full spectrum for each fluorophore enabling more parameters per study. After all, we always like to get more from less, right? Spectral unmixing is analogous to high-tech sortation process in a retailer warehouse where packages need to be sorted by zip code, size, weight, fragility etc. High-parameter cell sorting flow cytometry studies based on spectral unmixing allow scientists an increased depth of cell data to facilitate better understanding of immunological cell mechanisms, difference between samples, disease states and open new pathways for discovery. In a way, high-content studies provide more information than was previously possible.

Cell Sorter of A Different Kind

Though there are various methods of cell sorting, electrostatic droplet sorting at high-speed is a standard technique. Microtiter plate sorting is a powerful tool that combines advantages of flow cytometry single-cell resolution and droplet-based cell sorting. The Invitrogen™ Bigfoot Spectral Cell Sorter utilizes this technology with significant advances in plate deposition and calibration which gives it an edge over conventional cell sorting instrumentation. The spectral unmixing capabilities of the Bigfoot Spectral Cell Sorter allows you to expand your immunophenotyping reach with more experimental parameters. Typical cell sorters available today offer either conventional or spectral unmixing options.

Only The Bigfoot, with up to 9 lasers and 60 detectors, lets you migrate your existing experimental setup to spectral unmixing to expand your panels or, continue using conventional compensation. Both options add a new dimension of flexibility to your studies. It can sort a 96-well plate in less than 8 seconds thanks to multi-way plate sorting deflection capability. One may ask, but what about the errors? Well, innovations in plate deposition provide accuracy, recovery, and speed like never before. Technological improvements include a multitude of build-in sensors to monitor the entire system real time and high-speed custom field programmable gate array (FPGA) electronics.

Benefits of Choosing the Right Instrument

High parameter features such as user-configurable sort output, virtual sorting of multiple sort populations, integrated error monitoring, and jet-in-air sensing make the Bigfoot Spectral Cell Sorter one of a kind. High-performance and high-throughput, integrated safety, choice of conventional compensation or spectral unmixing, automated and easy to use wizard driven software, flexibility to support an array of cell types and applications are only some of the many features which makes the Invitrogen Bigfoot Spectral Cell Sorter a must-have for your laboratory or core facility.

Resources and Solutions for Flow cytometry

• Flow Cytometry Panel Builder – this online tool helps you design an effective reagent panel for your specific application on your conventional or spectral flow cytometer
• Flow Cytometry Protocols Handbook – protocols that fit your needs in flow cytometry ranging from sample preparation to numerous cell stimulation conditions, staining, immunophenotyping and data analysis strategies
• Immunology at Work Resource Center – practical guidance and protocols to help you get started quickly and confidently