Flourescence Activated Cell Sorting Core - Children's Hospital Los Angeles

Summary

The Fluorescence Activated Cell Sorting (FACS) core provides researchers with a powerful method to study and purify cells for wide applications in immunology, cell biology and other fields of biology.

Flow Cytometry is a technology used to make fluorescent measurements of special fluorochromes bound to or made by various cells as they travel in single file through one or more laser lines while confined by a laminar fluid. Common applications involve immunolabeling of extracellular or intracellular proteins with fluorochrome-conjugated antibodies, reactive dyes, or nucleic acid dyes. Fluorochromes are excited to emit various colors of light by the lasers. Data acquisition is achieved with specialized computer software directly interfaced with the cytometer.

Equipment/Services

2nd Floor FACS

The 2nd floor FACS core houses five cytometers, two analyzers and two sorters and a novel imaging flow cytometer (Imagestream). We use BD Biosciences and Amnis instrumentation.

FACScan (BD) Analyzer
Room 205
The three-color FACScan (BD) analyzer is a one-laser instrument, capable of collecting a maximum of seven parameters: two scatter, three colors (green, yellow-orange and red), plus two derived parameters. CellQuest Pro (v5.x) is the software driving this instrument.

FACSCalibur (BD) analyzer
Room 205
The four-color FACSCalibur (BD) analyzer is a two-laser instrument, capable of collecting a maximum of eight parameters: two scatter, four colors (green, yellow-orange, red and a second red), plus two derived parameters. CellQuest Pro (v5.x) is the software driving this instrument.

FACSAria-I (BD)
Room 203
The FACSAria-I (BD) instrument is equipped with three lasers, capable of detecting up to 13 colors, and is a high-speed cell sorter. The Aria-I is configured with BD Biosciences’ patented octagon/trigon optical bench and new-age flow cell, providing fixed alignment with stream in air sorting. Delivering four populations simultaneously to a purity frequently in excess of 99%, the FACSArias have been surpassing expectations. The Aria-I laser bench holds solid-state lasers, 488nm, 633nm, and 405nm, which are delivered to the flow cell via fiber optic cabling. Diva software (v6.x) is the software driving the instrument.

DiVa FACS Vantage SE (BD)
Room 203
The DiVa FACS Vantage SE (BD) has three lasers, a 488 nm Laser, a 635 nm, and the Innova I-302 tunable krypton laser with UV, Violet, Green*, Yellow or Red line capability. The Vantage collects up to 8 colors: 4 parameters (laser 1), two parameters (laser 2) and two parameters (laser 3). This is in addition to two scatter parameters as well as derived parameters. The optical system is operator-aligned and optimized daily for specific experimental needs. Designed for flexibility, many specialized assays including Calcium Flux and side population, are performed on the DiVa FACSVantage SE. Recently upgraded with a stand-alone air compressor, the DiVa FACSVantage is capable of ultra slow speed to high speed cell sorting. It can deliver four populations simultaneously to a purity frequently in excess of 99%. Diva (v5.x) is the software driving the instrument.
*New optics have been installed for this capability.

Imagestream 100 (Amnis)
Room 205
The Imagestream 100 (Amnis) is the newest addition to our cytometer line. This novel instrument is the first imaging flow cytometer, encouraging you to “Think outside the dot”. Loaded with three lasers (488nm, 633nm, 405nm) it will capture six (6) digital images (spatial resolution: 0.75NA, 0.5μm pixel size) of each cell, measuring morphology, brightfield and up to 4 parameters of fluorescence. The precisely controlled fluidics positions the cells in the plane of focus as they flow through the system smoothly and without tumbling. The spectral decomposition element splits the cell imagery into six spectral bands, one for each CCD camera. The unique time-delay integration tracks the motion of the cells and increases the signal 1,000 fold before image is recorded. Inspire (v3.x) software in used for data acquisition and IDEAS (v3.x) software is used for data analysis.

Analysis Stations
Rooms 203, 205, 206
For data acquired with 2nd floor instrumentation, analysis stations are available for your use free-of-charge. Software packages currently available include: CellQuest (5.x), FlowJo-Mac (v8.x), Flow Jo-PC (v7.x), VenturiOne (v3), Modfit (v3), FCS express and DIVA.

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3rd Floor FACS

The 3rd floor FACS Core has two research grade flow cytometers that use the latest in optical fibers to transmit photons of light from laser-excited dyes to highly efficient optical arrays.

LSR II Analyzer
(Room 301)

The LSR II from Becton Dickinson (BD) is an advanced analyzer equipped with 4 lasers emitting at 488 nm (blue), 633 nm (red), 405 nm (violet), and 355 nm (ultra-violet).

This system is dedicated for analytical use, and is able to detect up to 16 colors per cell simultaneously, allowing for highly complex cocktails of antibodies for immunophenotyping. Forward and side light scatter are also detected, for a total of 18 parameters. The system is able to detect multiple fluorescent protein colors (eg., GFP, RFP), and is compatible with multitudes of DNA stains and with new quantum dot technology.

FACSAria-I (BD)
Room 301

Analysis Stations
Room 301

Users may perform data analysis at the LSR-II dedicated computer free-of-charge, but only when it is not in use for acquisition (See appointment calendar for LSR-II acquisition schedule). Software packages currently available include: FlowJo-PC (v7.x), Multi-cycle, Diva.

Price List

Fee Schedule

Sorting	$125/hour
Specialized Pre-Sort Setup	$125/hour
Operator Assisted Data Acquisition	$125/hour
Training (Sorter training in not available)	$125/hour
Data Acquisition by a Trained User	$75/hour
Imagestream - Operator	$125/hour*
Imagestream - User	$75/hour

 *Note: For Childrens Hospital Los Angeles investigators, the first two appointments are FREE; Full Day Access Flat Fee: $500 (discuss with the director or manager for lengthy experiments)

Access to the FACS Core Facilities and services for non-Childrens Hospital Los Angeles researchers at USC and other institutions can be arranged. Interested researchers should contact the core directors about availability and rates.
 

Billing Policy

All appointments will be rounded to the nearest quarter hour and billed in quarter-hour increments. Investigators may be asked to sign in acceptance of sort appointment length. 

• 2nd Floor Billing
  On the 2nd floor, an accounting software program monitors user login times and will record actual use time on the analyzers. The analyzers are booked for data acquisition, however, you may analyze on the cytometer during your appointment time, but you will incur charges for this analysis time, data analysis is free of charge on the designated analysis stations.
• Third Floor Billing
  On the 3rd floor, appointments are quantified according to the time of acquisition of the first and last tubes, as recorded by the computer.  Analyzers appointments will be billed in quarter hour increments.

Appointments will be tallied monthly and submitted to The Saban Research Institute administration for billing to the Principal Investigators of Childrens Hospital Los Angeles.  The Flow Cytometry Core cannot be expected to be aware of any collaborations existing between the investigators, and therefore, will be billing according to the login account and user defined information.

• For Childrens Hospital Los Angeles investigators, a valid project number will be requested and kept on file.  Please bring new project numbers as needed. 
• For non-Childrens Hospital Los Angeles investigators, an invoice for services rendered will be emailed to the contact at the close of each month. Payments by purchase order number or check are to be sent to The Saban Research Institute, according to invoice details.

Appointment Policy

Sorter Appointment
Sorter appointment length is determined by the sort operator solely upon information given at time of appointment.  You will be asked for the following information:

• Desired final quantity of sorted cells.
• Frequency of the subpopulation to be sorted.
• Quantity of cells in initial population.
• Number of tubes which will be sorted.

Appointment length is based on the cell number of each sort specimen and the experimental design determines which cell sorter is used. In the end, you are billed for what you use, not for what we allotted time for. Appointments are accepted up to two weeks in advance and are finalized in the week prior to use.

Analyzer Appointment
For analyzer appointments, trained users and/or flow core personnel may schedule appointments, which:

• Are scheduled in half-hour blocks, with a maximum of two-hours for any one appointment.
• May be scheduled up to two weeks in advance.
• Must end by the scheduled time to maintain a courteous work environment.

Tampering with the appointment calendars is an unacceptable behavior for which offenders will be denied access to the Flow Cytometry Laboratory Resource. 

Analyzer Calendars

• Principal investigators working in The Saban Research Institute can make their appointments directly using our online calendars.
• Principal investigators from other facilities should contact the Core to set up an appointment. 

Cancellation Policy

We understand that the logistics of research experiments fluctuate and personal emergencies arise.  When those situations arise, please cancel your experiment as soon as possible to allow others access to the instrumentation by calling or emailing the personnel who manage the instrument you scheduled to use. 

Advanced Notice
Canceling an appointment, which would require the assistance from Core personnel, will require a minimum of 4 hours notice to avoid being billed at full rate for a minimum of one hour.  Morning appointments (beginning before noon) must be cancelled the previous evening by 5PM. 

There are occasions when the instruments go down, requiring unscheduled maintenance.  Any appointments cancelled due to down instrumentation will be not billed to investigators. In addition, if core staffing is unavailable for a scheduled sort, the investigator will not be billed.

Turn Around Time

Contact us for further information.

Abstract

Flow Cytometry is a technology used to take fluorescent measurements of special fluorochromes bound to or made by various cells as they are travel in a single file line while confined by a laminar fluid. Common applications involve immunolabeling of extracellular or intracellular proteins with fluorochrome-conjugated antibodies, reactive dyes, or nucleic acid dyes. Excitation of the fluorochromes is achieved with lasers. Data acquisition is achieved with specialized computer software directly interfaced with the cytometer. Listed alphabetically are applications supported by instrumentation at the Saban Research Institute: Absolute Counts, Apoptosis, Calcium Flux, Co-localization*, Cytometic Bead Assay (CBA) for up to 30 soluble cytokines or proteins per sample, DNA Cell Cycle Analysis, Flow-FISH for telomere length, Immunophenotyping for Surface, Intracellular and Nuclear proteins, Live cell sorting, PhosFlow, Rare Event Detection/Sorting, Reporter Molecules, Side Population, Single-cell Cloning, Single Color Apoptosis*, Sizing, Spot Counting for viral load*, Subcellular compartmentalization*, Synapse activity*,Translocation*, and Viability. *denotes applications specific to the Imagestream 100.

More information

Website

Visit the Website

Location

Smith Research Tower 4546 Sunset Blvd. 2nd Floor: Rooms 203 and 205, MS#62 3rd Floor: Room 301, MS #57 Los Angeles, CA 90027 Parking There is visitor parking in the hospital lot across from Smith Research Tower.

Contact Information

Collaborations | Consultations | Service Issues | Non-Standard Applications

Hisham Abdel-Azim, MD
Director
Telephone: (323) 361-5664
Fax: (323)906-8193
Email: habdelazim@chla.usc.edu

Michael Sheard, PhD
Director
Telephone: (323) 361-5664
Fax: (323)906-8193
Email: msheard@chla.usc.edu

Scheduling Appointments

Appointments | Training Questions

Ann George
Sort Operator
Telephone: (323) 361-5935
Email: ageorge@chla.usc.edu

Ewa Zielinska
Sort Operator
Telephone: (323) 361-5953
E-mail: ezielinska@chal.usc.edu

Jakie LIn
Sort Operator
Telephone: (323) 361-5455
Email: TSLin@chla.usc.edu