Instrumentation & Services
Instrumentation
Influxes (Jet-in-Air technology)
Optical Configuration (Influx-1)
Optical Configuration (ShadowFACS)
Download Optical Configuration (XLS)
The Salk core has 3 state-of-the-art BD InfluxTM cell sorters. These are jet-in-air design instruments uniquely suited for isolating delicate cells, and for high-speed sorting. “Influx-1” is equipped with 5 lasers, 11 fluorescent detectors. “Kirin Influx” is equipped with 4 lasers, 16 fluorescent detectors. “ShadowFACS” is equipped with 4 lasers, 13 fluorescent detectors. Additionally, ShadowFACS is equipped with a forward scatter (FSC) pinhole assembly that facilitates detection of dim FSC signals when performing small particle analysis. All Influxes have biosafety enclosures that enable BSL2 rated sorts. Several features of the Influx (including flow cell characteristics and use of low-noise pre-amp electronics) come together to enable excellent detection sensitivity and stable high-throughput sorting, while maintaining the integrity and viability of sensitive cell types. It is also remarkably fast and accurate at sorting into 384-well plates (~3.5min/plate for highly abundant targets).
The Influx has several nozzle sizes (70, 86, 100, 140-um) available to accommodate a variety of particle sizes and can sort up to 6 populations simultaneously. Cells may be collected into a variety of tube types or multi-well plates (e.g. 384 well).
FACSAria Fusion (Fixed alignment cuvette)
Optical Configuration (Fusion)
Download Optical Configuration (XLS)
The BD Biosciences FACSAriaTM Fusion is a high-end cell sorter. The Salk’s Aria Fusion is from the Special Order Research Product (SORP) line, with air launched high power lasers. The Aria design integrates fully fixed stream and optical alignment with a patented quartz cuvette flow cell and optical gel-coupled optics. While geared towards high sensitivity, complex flow cytometry, it remains easy to use, controlled by the same Diva software used by other BD Biosciences analyzers in the core. It is configured with 13 fluorescence and 2 light scatter detectors. This instrument is equipped with a full enclosure Class II Type A2 biosafety cabinet for sorting up to biosafety rating level BSL2.
The Aria Fusion has several nozzle sizes available (70, 85, 100, 130-um) to accommodate a variety of particle sizes and can sort up to 4 populations simultaneously. Cells may be collected into a variety of tube types or multi-well plates (e.g. 384 well).
CITING USAGE IN PUBLICATIONS: This instrument was acquired by a NIH Shared Instrumentation Grant (S10) award along with additional funding support from Larry and Carol Greenfield Technology Fund. In addition to the Salk’s Cancer Center grant NIH-NCI CCSG: P30 014195, please reference S10-OD023689 for reporting the use of this sorter.
BD FACS Symphony A3
Download Optical Configuration (XLS)
The BD FACS Symphony A3 is a high-parameter flow cytometer equipped with ultra-quiet VPX electronics, powerful lasers and highly sensitive photomultiplier tubes (PMTs). The A3 has options for running samples via tubes or the high throughput sampler (HTS) compatible with 96 and 384 well plates. It is configured with 5 lasers and up to 23 fluorescent parameters + FSC and SSC. Technical resources for this instrument include onsite stain index optimization and fluorescent channel spillover testing to ensure optimal generation of application settings which are then linked to daily CS&T calibration. The A3 runs on FACS Diva software and is located in the Parker Room.
LSRII-1 and LSRII-2
Download Optical Configuration (XLS)
The BD Biosciences LSR II is a configurable, high-end, analytical cytometer. A reliable platform well suited to complex polychromatic flow cytometry, the Salk’s Flow Core has two units equipped with 5 lasers, and the maximum 18 fluorescence detectors. Since the two are configured similarly, users are able to move easily from one instrument to the other. LSRII-2 is also equipped with a HTS (High Throughput Screening) module, which enables samples to be acquired in multi-well plates. The LSR II cytometer runs on BD FACS Diva acquisition software. Both units are located in the lower NE basement: LSRII-1 is located in the STEM Core, while LSRII-2 located across the corridor in the main FCCF Sorter Room.
Canto II
Optical Configuration (Canto II)
Download Optical Configuration (XLS)
The Becton-Dickinson FACSCanto II™ cytometer is an easy to operate 3-laser benchtop analyzer capable of detection of up to 8 fluorescent parameters and 2 light scatter parameters. The sample injection tube (SIT) features an automated flush function that rinses the probe between tubes to reduce sample cross contamination. The FACSCanto II™ runs on BD FACS Diva acquisition software. It is also equipped with a fluidics cart that holds all fluids necessary to operate, clean, maintain the instrument, as well as corresponding automated commands within the FACS Diva software to assist users in automatically starting up and shutting down the instrument in proper running buffer or fluidics shutdown fluids.
MACSQuant VYB
Download Optical Configuration (XLS)
The MACSQuant VYB is a simple to use compact benchtop flow cytometer equipped with 3 lasers (violet, yellow, and blue) and can detect up to 8-fluorescent parameters and 2 light scatter parameters. The MACSQuant VYB features semi-automated start up and calibration procedures, along with walk-away capabilities for automation. The VYB can acquire samples from a 96-well plate or from 5ml tubes (it is the only instrument in the core than can run a rack of 5ml tubes), seamlessly switching from one format to another with no calibration, priming, or other adjustment required by the operator. The VYB uses a robotic needle arm for sample uptake. Since this allows a highly precise volume to be taken up, cell concentration can additionally be determined at the time of acquisition (it is the only cytometer in the core equipped for direct cell counting). A built in self-shutdown feature allows users to automate cleaning from 5ml tubes after samples have been run before leaving the core; the VYB will perform shut down procedures before turning itself off (features a shutdown timer set by the core). The MACSQuant VYB has additional advanced capabilities for assisting with sample prep, for instance it can add reagents to samples prior to analysis (e.g. for cell activation assays).
- Practical Flow Cytometry– 4th Edition (Howard Shapiro)
- Flow Cytometry: A Basic Introduction (Wiki) (Mike Ormerod)
- BD Introduction to Flow Cytometry (on-line course)
- Selecting Reagents for Multicolor Flow Cytometry (Holden Maecker, Joe Trotter)
- Interpreting flow cytometry data: a guide for the perplexed (Herzenberg et al)
- Cytokine Encyclopedia
- Common phenotypic markers (Mouse and Human)
- Maturation markers reference
- Pathways database
Services
Using The Salk’s Flow Cytometry Core Facility
FCCF has 5 analyzers and 4 cell sorters. The Core facilitates access to Flow Cytometry technologies
through three main service categories: Staff Operated Cell Sorting, Cell Sorting Training, and
Analytical Flow Cytometry Training. The following sections guide users on how to access Flow Core
Services.
- Review FCCF’s current Core Access and Usage information on Salkland (link below).
- Review FCCF’s current training plan, complete any self study materials listed on Salkland.
- Contact the core to discuss your experimental needs and identify instrument(s) to train you on. At this time we can go over any protocols you will use to prepare samples for flow analysis. For maintaining instrument cleanliness, samples must include a DNAse I clean up (see Help File document on Salkland for more information and examples of where it can be conveniently included during sample preparation).
- Training: Orientation will be arranged (2 sessions, one to learn the processes, a second session to demonstrate proficiency). Upon completion, scheduler access is activated and we will schedule follow-up staff supported analysis session(s) as needed with the goal of helping you become independent. ➢ Click to go to Salkland (salk.edu/fccfaccess)
- Review FCCF’s current Core Access and Usage information on Salkland (link below).
- Review FCCF’s current training plan, complete any self study materials listed on Salkland.
- Contact the core to discuss your experimental needs and identify instrument(s) to train you on. At this time we can go over any protocols you will use to prepare samples for flow analysis. This will include going over what is needed to generate sort ready samples (e.g. DNAse I cleanup step to ensure a low viscosity high quality sort sample, and appropriate pH buffered sort buffers).
- Training: This takes place over a couple of sessions, in order to cover key skill blocks we require autonomous users to learn. We will arrange some “practice” sessions for you to demonstrate proficiency. Upon completion, access to the Google calendar will be shared. Follow-up staff supported analysis session(s) will be scheduled as needed with the goal of helping you become independent. ➢ Click to go to Salkland (salk.edu/fccfaccess)
- Before starting an experiment needing FACS (fluorescence activated cell sorting), read through FCCF guidelines on preparing for cell sorting. Be sure to include a DNAse I clean up step (or discuss alternatives with FCCF staff); this is to ensure you have a high quality, low viscosity sample appropriate for sorting.
- Submit a sort request form (on Salkland) BEFORE starting a time sensitive experiment. ➢ Click to go to Salkland (salk.edu/fccfaccess)