User information - Flow Cytometry
Tupper Hall, UC Davis campus
Becton Dickinson FACScan Flow Cytometer
This benchtop cell analyzer was originally equipped with a single 488nm laser and three photodetectors. In 2006, a second 635nm laser and two additional detectors were added through Opticore recharge funds, allowing investigators to detect five fluorescence signals per cell. Largely investigator-operated, it is heavily used for up analysis of cell phenotype, BrdU incorporation into DNA, DNA content for cell cycle studies, apoptosis, intracellular protein content and formation of oxidative products.
Becton Dickinson LSR-II Flow Cytometer
This 3-laser analytical cytometer combines digital pulse processing and fast analog to digital converters to acquire up to 17 fluorescence and two scatter signals per cell. High powered violet (50mW 405nm), blue (100mW 488nm) and red (50mW 635nm) lasers excite a variety of fluorochromes. The violet laser excites dyes such as Pacific Blue, Cascade Blue, DAPI, AmCyan, Pacific Orange, and 'Quantum Dot' semi-conductor nanocrystal dyes, including QD525, 565, 585, 605, 655, 705 and 805. The 488nm laser excites dyes such as FITC, Alexa 488, PE, PE-TexasRed, PECy5, PECy5.5, PECy7, and green fluorescent protein. The red laser excites APC, Alexa 647, Alexa 700, and APCCy7. In 2008, funding was provided to purchase additional photodetectors and a fluidics management system. In 2009, a new HP computer was installed to accommodate a major upgrade in acquisition and analysis software, DIVA 6.2. These upgrades have streamlined maintenance and quality assurance, allowing a large number of trained investigators to operate this cytometer independently.
Cytomation MoFlo Cell Sorter
This high-speed sorter (up to 25,000 cells per second) is one of the most sophisticated and flexible cell sorting systems in existence. It is custom-configured with three lasers providing multi-line visible and UV excitation. An Enterprise laser providing a 488nm line and a 352nm UV laser line, a tunable water-cooled Argon/Krypton mixed gas laser providing lines from UV to far red, and a water-cooled violet-enhanced Krypton laser giving dual 407 and 413nm UV excitation. In this set-up this instrument is capable of the simultaneous detection of up to 11 colors and two scatter parameters. Up to four populations of cells can be sorted simultaneously. A biohazard containment system is in place. The instrument has been used extensively for multicolor analysis, as well as cell sorting.
Compucyte iCys Laser Scanning Cytometer
This unique instrument combines quantitative cytometry and imaging to provide quantitative analysis of cells and tissues on slides or culture plates. It has an inverted Olympus IX71 microscope and a computer controlled stage that allows for automated, high-throughput scanning of stained tissue sections, tissue microarrays and live or fixed cultured cells. It is equipped with 405nm violet, 488 nm blue, and 633nm red lasers and uses four photodetectors and two photodiodes to collect fluorescence, light scatter and light absorption from specimens. Unlike confocal microscopy, the iCys collects total cellular fluorescence at a greater depth of field, permitting quantitation of total cellular fluorescence at each spatial location. Light scatter images can be combined with fluorescence signals to create unique 3D images combining fluorescence and cell morphology. A variety of dyes can be used, including DAPI, Pacific Blue, FITC, Alexa 488, Oregon Green, SYTOX Green, propidium iodide, PE, Cy5, Alexa 647, as well as histological stains. The iCys LSC has been used to quantify molecular markers identified by fluorescence in situ hybridization (FISH) and fluorescence immunostaining.
UC Davis Medical Center, Sacramento Campus
Becton Dickinson FACScan Flow Cytometer
This analytical cytometer is equipped with a 488nm laser to excite and measure 3 fluorochromes, typically FITC, PE and PECy5 or propidium iodide. It is used primarily for 1-3 color analysis of cells; most investigators are trained by Opticore staff to use this cytometer independently for cell cycle analysis, apoptosis assays, cell phenotyping and to quantitate GFP expression in transfected cells. It offers a simplified platform with CellQuest acquisition and analysis software for data analysis and display.
Becton Dickinson Fortessa Flow Cytometer
This is an advanced, special order analytical cytometer that is configured to use five intense, high-powered lasers and multiple photodetector arrays to collect fluorescence from a wide variety of fluorochromes. This cytometer is equipped with a 60mW UV (350nm), 100mW violet (405nm), 100mW blue (488nm), 100mW yellow (561nm) and 100mW red (640nm). Eighteen fluorescence and two scatter signals can be measured simultaneously. The UV laser allows for calcium flux assays (Indo-1 blue and violet) and DAPI excitation. Signals from violet excitation terminate in an array of six photodetectors that can measure a variety of Quantum Dots as well as Pacific Blue, AmCyan and Pacific Orange. The 488nm laser is used for GFP, FITC, Alexa488 and PerCPCy5.5. The newly-available 100mW 561nm yellow-green laser was chosen because this excitation beam most efficiently excites PE and PE tandem dyes such as PE-Texas Red, PECy5, PECy5.5 and PECy7. A highly powered red laser was chosen to improve excitation of red dyes. A high throughput sampling module increases sample loading and acquisition rates.
Becton Dickinson-Cytopeia InFlux Cell Sorter
This high-speed 5-laser cell sorter is located in Sacramento to provide operator-assisted cell sorting to researchers in Sacramento and from the Davis campus. This sorter is designed to maintain aseptic conditions, a crucial consideration prior to transplantation of sorted cells into immune deficient mice or in culturing sorted cells without antibiotics. Specifically, the inFlux is housed within a HEPA-filtered biocontainment cabinet that helps reduce atmospheric contaminants and was designed with simplified fluidic handling valves, exchangeable fluidics tubing, and disposable nozzle housings to eliminate microbial contamination niches and promote sterility. This sorter is sterilized with ethanol after each sorting session and is monitored for microbial contamination in antibiotic-free medium weekly. The inFlux sorter is equipped with five high-powered lasers to provide 100mW UV (355nm), 50mW violet (405nm), 200mW blue (488nm), 150mW green (532nm) and 50mW red (635nm) excitation beams and is configured to detect two-light scatter and 16 individual fluorescent markers. Recent upgrades include an Accudrop automated drop delay calibration system that will streamline cytometer setup prior to sorting and an advanced software package that offers improved sort setup and post-sort analysis tools.
Stratedigm S1400 Flow Cytometer
The S1400 analytical cytometer is located in Sacramento and offers compact design with simplified fluidics and optics systems to provide easy-to-use, cost-effective measurement of two scatter and eight fluorescence parameters. It is equipped with four smaller lasers, including violet (405nm), blue (488nm), yellow (561nm) and red (635nm) and uses highly sensitive photodetectors and a unique optical arrangement to measure standard fluorochromes such as Pacific Blue, FITC, PE, PE-TexasRed, PECy5, PECy7, APC and APCCy7, in addition to forward and side scatter. Many of the S1400's daily setup, standardization and quality control routines are automated, making the cytometer easy for a wide range of users who can be trained to operate the S1400 independently. Cell Capture also offers data analysis capabilities for summarizing and display.
It is your responsibility to read and understand the following information:
The UC Davis Flow Cytometry Shared Resource provides analytic flow cytometry and cell sorting services at locations in Davis and Sacramento. To allow smooth operation and fair access to the equipment for all investigators and to maintain equipment in proper working order operational policies and procedures must be followed. Key operational policies are highlighted below. Please see additional information regarding other operational procedures in the following paragraphs.
- Appointments for all instrument usage time must be scheduled.
- Operator assistance is required for cell sorting.
- Cell sorting appointments require a minimum of 24 hours advance notice.
- Analytic cytometers: operator-assisted appointments require a minimum of 24 hours advance notice.
- Approved, trained users may make appointments for self-operation same day.
- Evening and weekend appointments are authorized for trained, self-operated use only.
- Cell sorters and the 5 laser Fortessa analytic cytometer: minimum one hour
- All other analytic cytometers: minimum 30 minutes
- Training sessions are a minimum of two hours and will be charged at the assisted user rate. Users must supply a sample for training purposes.
- The ability to independently operate the cytometers will be at the discretion of laboratory personnel.
- Re-training: Self-operation privileges are earned and subject to approval by laboratory management. Re-training will be scheduled at the discretion of Shared Resource staff, and billed as assisted time.
Biohazard policy for analytic cytometers:
- All samples run on bench-top analytic cytometers must be fixed.
Decontamination and fluidics washing:
- Proper decontamination and cleaning of the fluidics tubing and flow cell must follow each use of the cytometer.
- Post-acquisition washing and cleaning procedures are considered normal run time and will be billed as part of the total appointment time.
- All users will wash the fluidics for a minimum of 15 minutes following each use of the cytometer; HTS users must add additional time for HTS cleaning.
Facility Operating Procedures and Rules of Conduct
The Facility operating procedures described in this section ensure safe, efficient and equitable functioning. Any violation of these procedures may result in termination of access privileges.
Two instruments are available for use with biohazards: the DakoCytomation MoFlo in Tupper Hall and the Cytopeia inFlux Cell Sorter in the Institute for Regenerative Cures. Any use of biohazards must be discussed with the laboratory manager before the application is approved. Facility personnel must be notified of potential biohazards prior to scheduling appointments and running samples. Users are required to dispose of all tubes and samples left after analysis by taking such materials back to their home lab in containers approved for safe transport.
All experiments must comply with UC Davis regulations for biosafety. All samples run on bench-top analyzers must be fixed. For sorting of biohazards, an amendment must be approved and filed with EH&S. Failure to meet facility biohazard requirements will lead to termination of access.
Operators and self-operation
The FACScan, XL, LSC, and LSRII are all available for operation by trained personnel, or training is available for self-operation. Training is available by appointment only, and will be performed using samples provided by the investigator. Laboratory personnel will determine when the investigator is able to self-operate. Keys to the facility are available for after-hours use.
Sorter self-operation is a major commitment and recommended only if you will be sorting for a significant period of time. Self-operator privileges to run the sorters can be obtained by completing training with the facility operator. Initial sorter training with the operator is followed by hands-on experience until sufficient skill is acquired to self-operate.
Self-operators are expected to be capable of normal operations without the facility operator's assistance, including startup and shutdown, sorting, analysis, sterilization and maintenance (nozzle clogs, etc.). Sorter training and ability is required prior to self-operation authorization. Self-operators must negotiate with the facility operator for help when running solo. Extensive reliance on the facility operator assistance leads to full charges. Self-operators can sign up for 15 minutes of facility operator time and self-operate for the remaining time. Equipment abuse will lead to self-operator privilege curtailment until problems are clarified.
Operator-assisted runs are scheduled Monday through Friday, 10 a.m. to 6 p.m. Assisted use times can be negotiated after hours. Signups must be made on the Instrument Scheduling calendar. If you sign up for assisted use, you must make sure that a staff member is available for assistance at that time. Valid users may schedule an instrument by selecting Instrument Scheduling from the menu.
Signup times are charged if not cancelled 24 hours before the run. If a cancellation occurs during the 24 hours prior to a run, the time becomes available for others and there is still a charge. If signup time is shifted or the facility operator makes an adjustment, the late cancellation charge may be waived.
A signup entitles the user to the specified time slot. A user with a reservation may insist that his or her samples be run within the specified period and may terminate the previous user's run. If equipment malfunctions or delays prevent usage, the user may request that charges be reduced accordingly. This strict schedule interpretation is intended to enable users and operators to meet obligations. Billing is for the actual time the instrument is in use (including the minimum described below). To avoid the reservation of instruments for unrealistically long periods, if more than an hour of a previously reserved slot remains unused, the user will be billed for the extra time.
Facilities running time policy:
- Reservations are charged if not cancelled 24 hour in advance.
- Users must reserve 30 minutes minimum on all machines, except for the Fortessa 5 laser cytometer and the cell sorters, which require one hour.
- An extra charge will be incurred for runs that extend longer than the signup time.
- The facility operator must be notified if equipment is not usable.
Total running time can be adjusted when delays are due to:
- Previous users
- Equipment malfunction
Nozzle clog delays are caused by clumpy samples or excessive boost duration and are considered chargeable time. If the previous user left a clog or excessive time is needed to clear the flow-cell, running time adjustment will be made
Accounts are billed monthly through the Department of Medical Pathology. Billing questions should be directed to Bridget McLaughlin. A DaFis recharge number is required for facility use.
Facility users are responsible for all data archival.
Users should report problems at the Facility immediately to:
- Report instrumentation problems to Bridget McLaughlin.
- Director Dr. Barbara Shacklett is the final arbiter for problems that arise at the facility.
Holden T. Maecker, Tom Frey, Laurel E. Nomura, and Joe Trotter.
Selecting Fluorochrome Conjugates for Maximum Sensitivity Cytometry Part A, 62A: 169 - 173 (2004)
Tung JW, Parks DR, Moore WA, Herzenberg LA, Herzenberg LA.
New approaches to fluorescence compensation and visualization of FACS data. Clin Immunol. 2004 Mar; 110(3): 277-83.
Roederer M, Darzynkiewicz Z, Parks DR.
Guidelines for the presentation of flow cytometric data. Methods Cell Biol. 2004; 75:241-56.
Current Protocols in Cytometry
J. Paul Robinson and Zbigniew Darzynkiewicz, Editors
John Wiley & Sons, October 1997
In Living Color: Protocols in Flow Cytometry and Cell Sorting
Editors Rochelle Diamond and Susan DeMaggio, Springer-Verlag, 2000
Flow Cytometry: First Principles
Alice Longobardi Givan, Wiley-Liss, New York, 1992
Antibodies and Reagents
Calibration Standards and Controls
Compensation, Antibody Conjugation, Fluorochromes
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