High-throughput, high-parameter cell sorting with integrated biocontainment

Designed by cell sorting experts for today’s most challenging cell sorting applications, the Bigfoot Spectral Cell Sorter combines performance, safety, and ease-of-use to fit seamlessly into the workflow of both individual labs and core facilities. Bigfoot allows spectral unmixing or conventional compensation for high-parameter, high-throughput cell sorting experiments for maximum flexibility.

 


High performance and throughput

With sort rates exceeding 70,000 events per second (eps) and analysis rates of more than 100,000 eps, the Bigfoot Spectral Cell Sorter is lightning fast. The sorter is capable of 6-way sorting into tubes, multi-way sorting into 96-well and 384-well plates, or straight-down sorting into 1536-well plates. In addition, virtual 18-way sorting allows researchers to separate multiple populations from a single sample or different samples in sets of 6 populations. For bulk sorts, the InfiniSort capability lets you perform multiple tube or plate sorts in sequence.

 

The Bigfoot Spectral Cell Sorter can sort a 96-well plate in less than 8 seconds, and a 384-well plate in less than 11 seconds

Plate sorting is not only fast, it’s remarkably accurate. Using the HRP method, with visual confirmation of droplet deposition through the colorimetric conversion of TMB substrate, we have shown that a single droplet can be sorted in small volumes into both 96-well and 384-well PCR plates with 100% targeting accuracy. Our white paper explains the engineering innovations we have developed to obtain such fast and accurate sorting.

96 well plate option for cell sort delivery
384-well plate option for cell sort delivery

Accurate droplet deposition.  Images show plates following droplet event sorting of beads suspended in horseradish peroxide (HRP) solution into 3,3’,5,5’-tetramethylbenzidine (TMB) substrate. If TMB turns blue, it indicates accurate deposition. (Left) In a 96-well plate, wells in rows A–D received 4, 3, 2, and 1 droplet respectively, as evidence by blue gradient from strong to pale. Wells in row E received no droplets (confirmed by no color change), while rows F–H received 1 droplet per well. (Right) In a 384-well plate, odd rows (starting with A) received one droplet resulting in a blue hue, while even rows (starting with B) received no droplets and were colorless.

 


Integrated biocontainment and aerosol management

The Bigfoot Spectral Cell Sorter features an integrated biocontainment enclosure which provides personnel and product protection similar to a Class II biosafety cabinet.

 

Test procedures and criteria laid out within NSF49 and EN12469 can be utilized to demonstrate performance. The custom-designed enclosure protects operators and samples from aerosols without compromising high-parameter sorter performance or impacting workflow. A separate aerosol management system meets ISAC guidelines for cell sorters.

Integrated biosafety enclosure and aerosol management

An adjustable sash maintains containment while allowing easy access to the nozzle and sample/sort areas, assuring constant user protection. A separate dedicated aerosol management system (AMS), HEPA filter, and software provide containment warnings and increase flow if a clog is detected.

Bigfoot Cell Sorter with onboard computer and integrated biosafety cabinet

Thoughtful design has converted biocontainment from a cumbersome add-on to an integrated part of the Bigfoot instrument and user workflows. The adjustable sash can easily be raised or lowered for safe access to the sample area or sorting nozzles. An internal control panel in the sample area limits the need for the operator to reach in and out of the containment field. Air flowing through the cabinet is exhausted either into the laboratory or via an optional canopy connection, through an external exhaust system.


Choice of conventional compensation or spectral analysis

All Bigfoot Spectral Cell Sorter configurations function beautifully as conventional cell sorters, applying compensation to account for spectral spillover from one fluorophore (color) into another fluorophore’s detector. The Sasquatch software builds and applies up to a 60 x 60 compensation matrix (using all 60 detectors) for high-color experiments. This makes migrating existing conventional flow cytometry experiments to your Bigfoot instrument easy, without the need to change protocols or analysis methods. You still gain the benefits of high-throughput, high-performance, easy-to-use cell sorting with integrated biocontainment.

 

In addition, most Bigfoot configurations enable spectral flow cytometry, using spectral unmixing instead of compensation to separate the signals from different fluorophores. By analyzing the full spectrum of signal from every fluorophore in your experiment, spectral analysis allows you to use fluorophores with similar signals in the same experiment, increasing the number of parameters you can analyze. This is especially useful for deep immunophenotyping, biomarker screening, and other applications.

 

With Bigfoot spectral configurations, you can run both conventional and spectral experiments on the same sorter, and convert experiments from conventional to spectral when you need more parameters.

Spectral flow cytometry

One important benefit of spectral optics and analysis is a dramatic increase in the number of parameters (fluorophores) that can be detected in a single experiment. The Bigfoot Spectral Cell Sorter can be configured with up to 9 lasers and 60 detectors, supporting spectral sorting and analysis of cell populations in expansive panels. For example, the figure shows a combined spectral signature graph for multiple fluorophores, showing not only how each fluorophore’s signature is unique, but also where additional fluorophores might be added.

Spectral signature graph.

In Sasquatch (SQ) Software, you can see a visual representation of the selected panel of fluorophores. The graph provides a clear picture of where additional fluorophores can be added without overlapping with the current selections. A specific emission curve can be shaded by highlighting it in the Selected Fluorophores list.

Spectral signatures of over 50 different fluorophores processed by SQ software

A second advantage of spectral unmixing and analysis is that autofluorescence can be treated as just another fluorophore with its own spectral signature. Unlike in conventional flow cytometry, autofluorescence does not limit dye options and can be assessed or eliminated depending on the needs of the experiment.

 

For a detailed examination of spectral flow cytometry hardware, optics, analysis, advantages, and disadvantages, see the article Spectral Flow Cytometry Fundamentals in our Flow Cytometry Learning Center.

Real-time spectral unmixing

Some spectral cell analyzers perform unmixing after all cells are acquired, but this is inadequate for cell sorting, since cells are sorted as they are acquired and processed. The Bigfoot Spectral Cell Sorter performs unmixing in real time and applies the results to the sort. A massively parallel, pipelined data processing architecture eliminates hard aborts that can result in lost events with higher event rates. The high resolution of the unmixing algorithm allows accurate setting of sort gates maintaining the purity of the sorted cell populations.

Sectral sorting applications

The Bigfoot Spectral Cell Sorter is especially well suited for sorting applications that require numerous parameters or high throughput.

Immunology

 

Immunophenotyping parameters have increased as researchers study cell subsets beyond major immune subgroups and effector and memory cells

Cell and gene therapy research

 

Therapeutic candidates such as stem cells and CAR-T cells must be efficiently sorted both pre- and post-manipulation

Gene editing

 

Sort cells of interest prior to editing and collect populations of cells edited by CRISPR or other methods

Gene sequencing

 

With output flexibility, the Bigfoot Spectral Cell Sorter can sort single cells directly onto a 10X Genomics chip


Automated and wizard driven software for ease of use

Advanced automation and software wizards allow operators with all levels of knowledge to use and control the system with confidence. For example, the formation and timing of cell-containing droplets—the drop delay between interrogation of the cell and its break-off from the stream to be sorted—is crucial to ensure that the correct cells are sorted into the correct tubes. Integrated control systems automatically ensure optimal droplet formation persistence and position stabilization over time, maintaining accurate and stable drop delay through the day.

To streamline workflow and reduce user errors, automated startup, stream calibration, quality control, and drop delay simplify sort setup and let you program the system to be ready when you are. The electronics automatically configure optimal laser delay for different nozzle sizes and pressures. If a bubble is detected, the system notifies the operator and stops the sort to preserve precious samples. Integrated temperature control (from 4–37°C) and agitation maintain the integrity of sample input, while output temperature control maintains the viability of sorted samples.

 

Sasquatch (SQ) software presents a clean user interface with intuitive workflow, simplifying acquisition, analysis, and sorting. An advanced fluorophore selector helps with design for both standard and spectral experiments and warns of potential conflicts. The experiment wizard assists in running controls, identifying potential issues, and applying the unmixing algorithms.

 

Analysis features include overlays, color-gating, backgating, index sorting, and ratio plots, and the software generates FCS (flow cytometry standard) files compatible with other analysis software packages. The software also produces clear reports on system usage, quality control, trending, and a sort summary.

System health check

Sasquatch software continuously tracks system status and health. You can monitor and control the system from the attached workstation or remotely. Automated startup allows the Bigfoot Spectral Cell Sorter to turn itself on and run QC protocols before anyone arrives at the lab.

SQ health screen

Sort pre-check

The software maintains a checklist of all tasks required before sorting and displays a visual indicator of their status. A wizard guides you through the steps of making sure everything is ready to sort.

Cell Sort precheck screen

Camera surveillance

The Droplet camera shows droplets and break-off in real time. Other cameras allow you to monitor media and sort streams.

Droplet camera

Complexity index

As you add fluorophores to your panel, the complexity index (rightmost column) estimates how complicated it will be to unmix the spectral signatures. Ideally, each added fluorophore will minimally increase the index value.

Complexity assessment

Spectral similarity matrix

SQ software computes the spectral similarity between all pairs of fluorophores in your panel, then flags the ones that cause an increase in complexity and thus may be more difficult to spectrally unmix.

spectral matrix to understand overlap for specific fluorophores

Panel builder

Our advanced, updated online tool guides you through flow cytometry panel design for each experiment using either conventional or spectral analysis. The tool offers a simplified, customizable interface, valuable for all experience levels.

Multicolor antibody design tool for flow cytometry and cell sorting

Flexibility to support a range of cell types and applications

The Bigfoot system offers sampling from six input positions that automatically detect the tube size (1.5, 5, or 15 mL) based on the adapter used. The multi-sample loader allows the controls for a 5- to 10-color experiment to be loaded in just one or two sets. The multi-tube loading capability is especially efficient for studying 20 or more fluorescence parameters, which is increasingly common in flow cytometry. All sample-related subsystems, including the multi-sample loader, are contained inside the fully integrated biosafety cabinet for optimal safety, efficiency, and performance.

Six-position multi-sample loader

The Bigfoot Spectral Cell Sorter sample loader has six input positions from which you can load multiple samples for sorting and analysis. To ensure proper loading, the instrument detects the adapter type as the loader moves from sample to the next.

Various tubes can be used to load samples

One sample input location is reserved for calibration beads, eliminating the need to retrieve beads from a refrigerator, dilute, and prepare a tube for daily setup. An onboard wash station performs a complete wash and backflush cycle in 8 seconds, resulting in extremely low carryover between samples. When a sample’s volume runs low, the end-of-sample bubble detector responds within 50 ms to stop sampling, wash the probe, and move to the next tube without user interaction.

 

On the sort output side, the Bigfoot Spectral Cell Sorter’s jet-in-air sensing is gentle with fragile cells to maximize viability at high sort speeds without reducing yield. Precise single-droplet targeting ensures unprecedented accuracy, recovery, and speed down to single-cell sorting. Configurable sort output holders include 1.5, 5, 15, and 50 mL tubes, microwell plates up to 1,536 wells, microscope slides, and 10x Genomics chips. A swap tip wizard simplifies setup of 70, 100, 120, and 150 μm nozzle tips.


Streamlined design

Designed for today’s cell sorting applications from simple to complex, the Bigfoot Spectral Cell Sorter is loaded with thoughtful design features that increase flexibility, boost performance, and streamline your workflow. For example, calibration beads are kept ready for use onboard the sample loader, eliminating the need to prepare a new calibration solution each day. (Simply replace the spent vial with a new one about once a month.) Within the biocontainment field there is ample deck space for sample racks and plates as well as access to a built-in sample vortex mixer, tube rack, and biohazard bag. This allows the operator to complete many routine tasks without breaching the safety barrier.

With multiple cameras and sensors throughout, the system monitors system health, provides feedback on all subsystems, and logs status for proactive troubleshooting. Built-in bubble detection automatically stops sampling and notifies the operator when the sample tube is nearly empty, reducing dead volume. The system tracks the volume of sorted samples and can change collection tubes automatically when full.


Lab or core facility friendly

While the Bigfoot Spectral Cell Sorter is designed to fit the workflow of an individual laboratory, it is also well suited as a cell sorting workhorse in a flow cytometry core facility.

 

You don’t have to take our word for it. In this video, three core facility managers who participated in our beta test program discussed their experiences bringing the Bigfoot Spectral Cell Sorter into their labs. They shared their insights about how the Bigfoot system has earned a place among the cell sorters they manage due to its performance, automation, ease of use, small footprint, low noise level, and flexibility. “I call it the Swiss army knife of cytometers,” said Rachael Walker of the Babraham Institute outside of Cambridge, England.

Although flow cores are increasingly reliant on self-serve instruments, high-end cell sorters have not been easy to convert to end-user operation. “The amount of time and expertise it takes to run [a competitor’s high-end] instrument is beyond the majority of our user base,” said Patricia Rogers of the Broad Institute in Cambridge, MA. “They just don’t have six months to learn how to do laser alignment and verticality like we do.” By incorporating quick startup, automation, software wizards, and easy training, added Rogers, the Bigfoot Spectral Cell Sorter has proved that a high-end sorter can indeed be operated successfully by end users, freeing staff to focus on new technology and assay development.

“The automated startup, alignment, and QC on the Bigfoot Sorter is amazing—17 minutes from start to finish. Automated startup saves us at least an hour a day. That is an hour’s more sorting that I can charge for in my core facility. That equates to 20 hours a month more capacity and more income into my facility. The Bigfoot Sorter has been built for the core facility.”

Rachael Walker,

Head of Flow Cytometry

Babraham Institute, Babraham, Cambridgeshire, England


For Research Use Only. Not for use in diagnostic procedures.