Flow Cytometry

Flow Cytometry

Overview

The LJI Flow Cytometry Core Facility is one of the largest flow cytometry cores in the San Diego area. The core facility is a full service flow cytometry laboratory that provides investigators with state-of-the-art equipment along with the necessary expertise and services to support cutting-edge research. The core facility offers a wide range of routine and custom flow cytometry, mass cytometry, cell sorting services, data analysis, strategic planning for novel assays, user training as well as full scale research collaborations. More than fee-for-service providers, flow cytometry core staff members are personally invested in the success of experiments and consider themselves temporary extensions of individual labs. The flow cytometry facility has extensive experience in daily instrument calibration and maintenance, multicolor panel design, immunophenotyping, intracellular and intranuclear staining.

Instrumentation

Digital Analyzers

LSRII-1 / LSRII-2 / LSRII-3

The BD LSRII is a benchtop analyzer capable of multi-color acquisition through digital electronics for increased sensitivity. The LSRII uses Diva acquisition software and features Auto-Compensation and overall software flexibility. Each LSRII has a slightly different optical configuration equipped with up to 4 lasers, and can detect up to 13 fluorescent detectors and 2 scatter parameters.

LSR Fortessa

The BD LSR Fortessa is located in the RNAi facility. Users with RNAi projects are given priority scheduling for this instrument. The BD LSR Fortessa is a benchtop analyzer capable of multi-color analysis through digital electronics for increased sensitivity. The LSR Fortessa uses Diva acquisition software and features Auto-Compensation and overall software flexibility. This instrument is equipped with 5 lasers, 18 fluorescent detectors and 2 scatter parameters. The LSR Fortessa is also equipped with the HTS (High Throughput Screening) option, allowing for acquisition from a 96-well or 384-well plate.

FACSCanto-II

The BD FACSCanto-II is a benchtop analyzer capable of multi-color analysis through digital electronics for increased sensitivity. The FACSCanto-II is equipped with 3 lasers allowing detection of up to 8 fluorescent markers and 2 scatter parameters. This instrument has a fluidics cart that holds all fluid tanks necessary to operate and maintain the instrument. It also features a SIT flush cycle between tube changing to reduce cross-contamination between samples. The FACSCanto-II is also equipped with the HTS (High Throughput Screening) option, allowing for acquisition from a 96-well or 384-well plate.

Accuri C6

The Accuri C6 is a compact and simple cytometer equipped with 2 lasers, 4 fluorescent detectors, and 2 scatter parameters. This instrument has pre-optimized detector settings so that users do not need to adjust any settings. This instrument is also capable of absolute cell counting.

Analog Analyzer

FACSCalibur

The BD FACSCalibur is a benchtop analyzer that is equipped with 2 lasers, capable of detecting up to 4 colors. This instrument is an analog analyzer that runs off of CellQuest Pro acquisition software.

Cell Sorters

FACSAria-1 / FACSAria-2 / FACSAria-3 / FACSAria Fusion

The FACSAria-1 sorter was originally a BD FACSAria-I cell sorter, but was recently upgraded to a FACSAria-II cell sorter (FACSAria-IIu). The upgrade included a complete and improved fluidics pathway change and additional safety features providing enhanced aerosol containment for BSL-2 sorting. This instrument is equipped with 3 lasers: a blue 488nm, a red 633nm, and a violet 405nm. The optical configuration allows multi-parameter detection of up to 9 fluorescent parameters and 2 light scatter parameters. The FACSAria-1 is capable of sorting through up to 50 million cells per hour (dependent on cell type), with up to four populations simultaneously. The populations of interest can be sorted into 0.5 mL/1 mL eppendorf tubes, 5 mL tubes, or 15 mL conical tubes. Populations can also be sorted directly into multi-well culture plates and PCR tubes, including: 12-well, 24-well, 48-well, 96-well, and 384-well.

The FACSAria-2 sorter is the original model FACSAria-I cell sorter that as been upgraded with a FACSAria-II flow cell. It is equipped with 3 lasers: a blue 488nm, a red 633nm, and a violet 405nm. The optical configuration allows multi-parameter detection of up to 9 fluorescent parameters and 2 light scatter parameters. The FACSAria-2 is capable of sorting through over 50 million cells per hour (dependent on cell type), with up to four populations simultaneously. The populations of interest can be sorted into 0.5 mL/1 mL eppendorf tubes, 5 mL tubes, or 15 mL conical tubes. Populations can also be sorted directly into multi-well plates, including: 12-well, 24-well, 48-well, 96-well.

This FACSAria-II model is the second generation of FACSAria cell sorters. The FACSAria-3 is equipped with 4 lasers: a blue 488nm, a red 640nm, a violet 405nm, and a green/yellow 561nm. The optical configuration of this instrument allows detection of up to 13 fluorescent parameters and 2 light scatter parameters. The green/yellow laser is optimal for excitation and detection of red and orange fluorescent proteins, such as mRFP/DSRed/mCherry. The populations of interest can be sorted into 0.5 mL/1 mL eppendorf tubes, 5 mL tubes, or 15 mL conical tubes. Populations can also be sorted directly into multi-well culture plates and PCR tubes, including: 12-well, 24-well, 48-well, 96-well, and 384-well. This instrument also runs off of the latest version Diva 8.0 software; the main advantage is that it runs off of Windows 7 and features Index Sorting. Index Sorting is available for single cell plate sorting and gives you a staining profile analysis of each cell in each well.

IMPORTANT: The BD FACSAria II was funded by NIH equipment grant S10RR027366. Please reference this award in any publications that utilize this equipment.

*The BD FACSAria-1, FACSAria-3 are properly contained for BSL-2 level sorting that does not require use of a Biosafety Cabinet, i.e. normal healthy donor.

Please see Biohazard Info for infectious/BSL-2 cell sorting.

The BD FACSAria Fusion is the newest model of the FACSAria family. This sorter is the latest addition to the core facility, installed in June 2015. This instrument is completely contained inside of a Baker Biosafety Cabinet and should be used for any samples potentially infected with BSL-2 pathogens that require BSL-2 + BSL-3 safety precautions (i.e. LCMV, HCV/HIV, non-human primate). This instrument is equipped with 4 solid-state lasers: a blue 488 nm, a violet 405nm, a yellow/green 561nm, and a red 642 nm laser. The optical configuration allows detection of up to 16 fluorescent parameters and 2 light scatter parameters. The populations of interest can be sorted into 0.5/1 mL eppendorf tubes, 5 mL tubes, 15 mL conical tubes, and 50 mL conical tubes. Populations can also be sorted directly into multi-well culture plates and PCR tubes, including: 12-well, 24-well, 48-well, 96-well, and 384-well.

*Priority will be given to the sorts that require the Biosafety Cabinet.

CyTOF Helios Mass Cytometer

The CyTOF Mass Cytometer was purchased in 2014 through the High-End Instrumentation (HEI) Award (S10). This is the first mass cytometer purchased by a non-profit in San Diego that will provide services to external users. Mass Cytometry allows for single-cell analysis of 40+ parameters without the use of fluorescence or the need for compensation, and theoretically has the capability of measuring over 100 parameters simultaneously. The CyTOF is based on an inductively coupled plasma mass spectrometer (ICP-MS) that uses stable isotopes of earth metals, typically lanthanides, as reporters. A purified single metal isotope is used to tag antibodies and these conjugates are detected by ICP-MS. This novel technology provides a new approach to high-dimensional cytometry.

In mass cytometry, cells are stained with antibodies conjugated to metal isotopes. Cells are also stained with iridium-conjugated DNA intercalators for initial detection of cells, and stained with intercalators for detection of viable cells. After labeling, the cells are injected and nebulized into single-cell droplets and introduced into a high-temperature plasma (~7000 K), which vaporizes the sample, creates atomic ion clouds that are pulled into the high vacuum of the mass spectrometer. The ion cloud has a lifespan of ~300 μsec and is detected by TOF-MS (time-of-flight mass spectrometry), and single cells can be resolved at an acquisition rate of <1000 cells/s. Each reporter is measured and quantified and data is recorded as a FCS file (Flow Cytometry Standard). CyTOF Services offered: custom panel design, access to LJI Antibody Bank (commonly used markers), custom antibody conjugation, user and staff-assisted sample acquisition, advanced data analysis with clustering algorithms (in development with Bioinformatics).

IMPORTANT: The CyTOF 2 mass cytometer was funded by NIH equipment grant S10OD018499. Please reference this award in any publications that utilize this equipment.

FlowJo Analysis Software

FlowJo Analysis Software is used for flow cytometry data analysis. LJI has a site license, which allows each user to download the software onto any LJI computer and to obtain a license key.

Cytobank Enterprise

Cytobank is a cloud-based platform used for high-dimensional flow and mass cytometry data analysis. Visualization tools include: Sunburst, Dose Response, Heat Maps, SPADE, viSNE, and dot and histogram overlays.

Rates

  2018 Internal Rates 2018 External Rates
FACS Analysis (per hour)
LSRII, LSR Fortessa or FACS Celesta (User-operated) $35.00 $66.15
LSRII-3, FACS Canto-II (User-operated) $25.00 $47.25
Accuri C6 (User-operated) no charge $47.25
Cell Sorting (per hour)
FACSArias (Staff-operated) $65.00 $122.85
FACS Melody (Staff-operated) $65.00 $122.85
FACS Arias or FACS Melody (User-operated) $55.00 $103.95
Mass Cytometry Instrument User (per hour)
CyTOF Helios (Staff-operated) $70.00 $264.60
CyTOF Helios (User-operated) $55.00 $207.90
CyTOF Services/Reagents Costs
Conjugation Reagent Kit (cost per reaction) $100.00 $378.00
Custom Antibody Conjugation (cost per reaction - user provides purified antibody) $120.00 $453.60
Antibody Bank Reagent - Antibody, Fluidigm (100 tests/cost per test) $4.00 $15.12
Antibody Bank Reagent - Antibody, Fluidigm (50 tests/cost per test) $8.00 $30.24
Antibody Bank Reagent - Common Core Stock, In-house conjugated (cost per test) $3.00 $11.34
Barcoding Reagent (Pd conjugated, per sample) $50.00 $189.00
Flow & Mass Cytometry Training/Other services
LSRII-1, LSRII-2, LSR Fortessa, FACSCelesta (session = charge for instrument use per hour) $35.00 $66.15
LSRII-3, FACSCanto-II (session = charge for instrument use per hour) $25.00 $47.25
Accuri C6, FACSCalibur (session = charge for instrument use per hour) no charge $37.80
FACSMelody (session = session for instrument use per hour) $55.00 $103.95
Technical assistance $60.00 $226.80

Resources

FluoroFinder – Panel Design Tool

A core resource used at leading flow cytometry research facilities.

Multicolor Flow Cytometry

BD - Intro to Flow Cytometry

Invitrogen - Fluorescence Tutorials

BD Fluorescence Spectrum Viewer

Fluorescence SpectraViewer

Flowjo - The Daily Dongle

BD - Basics of Sorting