- Training
- Scheduling and Cancellation
- Data Backup
- Guidelines for Using Analyzer
- Guidelines for Using High-Speed Cell Sorter
- Guidelines for Using ImageStream
- Violations and Fines
The Unified Flow Cytometry Core is the centralized FACS facility for the Oakland Campus of the University of Pittsburgh School of Medicine. It is equipped with two biocontained sorters, one non-contained sorter, and six analytic instruments. In addition to the standard blue, red and violet lines, instruments are equipped with the green, yellow-green and/or UV lasers. The sorters are: a biocontained BD Aria IIU (5 laser, 17 detector), biocontained BD Aria II (5 laser, 17 detector), and non-contained BD Aria IIU (3 laser, 12 detector). The flow analyzers are: BD LSRFortessa (5 laser, 18 detector), BD LSRII (5 laser, 17 detector), BD LSRII (5 laser, 17 detector), BD LSRFortessa (4 laser, 15 detector), BD LSRFortessa (4 laser, 15 detector), Attune NxT with CytKiK MAX auto-sampler (4 laser, 16 detector), Amnis ImageStream imaging cytometer (6 laser, 10 detector), Canopy ZellScanner ONE Chip Cytometer, and two Cytek Aurora spectral cytometers with up 64 fluorescent channels. The core is approved for specialized BSL2+ sorting.
New user training for analyzer and Image Stream (update 7/6/2015)
All new users are required to meet the Core staff and fill out the New User Form through the website and notify the core at: flowcore@pitt.edu
Users who have never operated the existing models of the instruments in the Core must be trained. Users who request training on the analyzers with no Flow Cytometry experience are required to read the introductory material online (http://www.bdbiosciences.com/services/training/itf_launch.jsp) and the BD FACS Diva Basic Experiment Guide before training.
Two training options are provided:
1. Training provided by Core staff: Two sessions will be provided. For the first session, Core staff will prepare the samples and teach general requirements, policies, basic hardware and software features. Group training may be arranged. For the second session, the trainee brings his/her own samples and practices the steps taught in the first session. This is a one-on-one session.
2. Training provided by the experienced users: The trainee can work with an experienced user for as many times as needed. Once the experienced user thinks the trainee can handle the instrument/software independently, the trainee will then schedule an appointment with Core staff to go over our general requirements and policies.
Users who come from other departments/institutes with experience operating similar models of the instruments used in the Core are not required to participate in further training. However, he/she must schedule an appointment with Core staff to go over our general requirements and policies.
Super user training for cell sorter
In order to facilitate the maximum productivity of our users, the Unified Core will make it possible for individual users to operate the cell sorters under certain circumstances. Such individuals will be deemed “super users.” In general, a trained super user should schedule the sorting during nights, weekends and holidays. During regular work hours, Core staff has the priority to use the cell sorters. All sorting must be done by available Core staff at regular rates. Given the lack of need for an operator for unattended sorting by super users, special, lower rates will be established.
A user can request to be trained as a super user if they meet all of the following three criteria:
1. 1 year or more of hands-on Flow Cytometry experience.
2. A demonstrated history of using both analyzers (hands-on, self-operated) and cell sorters (user-provided samples sorted by Core personnel) for the past 6 months in either the United Flow Core, our partnering cores, or an external equivalent facility.
3. A need for at least 3 hours/week of non-biohazardous cell sorting during the off-work hours (nights, weekends and holidays).
A written request should be sent to flowcore@pitt.edu outlining how the user meets the above criteria. The request will then be considered by Core Leadership.
Multiple training sessions will be arranged with Core staff on a first-come first-served basis to learn the instrument’s set-up and shut down procedures, the sorting set-up for an experiment, and basic troubleshooting. The time needed for training will be based on individual experience and learning. Training time will be billed at per-hour rates (same rate as regular cell sorting). Certification for sorting independently will be at the sole discretion of Core staff. Please note that based on how busy the facility is there may be a limited number of training slots available at any given time.
If a user has operated a similar model or even the same model of cell sorter in another institute, he/she still required to be trained and demonstrate proficiency and knowledge of the procedures to Core staff. In this case, the training time can be dramatically shortened if such proficiency is demonstrated successfully.
Scheduling
Trained users must schedule experiments on the individual instrument calendar by logging into www.schedulebook.com.
Users should try not to overbook time because unused time will be charged. If the difference of the time scheduled and the time used is greater than 90 minutes, the remaining time must be charged to no NIH funded sources (02 or 04 accounts).
During business hours (9am-5pm) users are required to limit reservations to 5 hours. Longer sessions can be booked after hours or on weekends.
If the user is unable to complete his/her samples within the allotted time and the user scheduled after needs to use the instrument, the previous user must leave.
To analyze samples containing potential bio-hazards without fixation, such as unfixed human, non-human primate or virally-transduced or infected cells, etc., users must schedule using the instrument (STI Fortessa) within a containment hood, which is located in south BST Room S756 or run them on the Aurora by folowing the BSL2 requirements from EH&S. Samples containing known biohazard or infectious agents which are above BSL2 MUST be fixed in a 0.5-2% paraformaldehyde solution for at least 30 minutes before acquisition.
For cell sorting or assisted analysis, first time users are required to meet Core staff. The subsequent appointment can be made by phone or email.
Changing or cancelling an appointment
Any changes or cancellations of appointments should be done at least 24 hours before the scheduled time to avoid a charge. The Core cannot accept excuses for same day cancellations other than cytometer malfuntion.
If something unexpected comes up during the same day of an appointment and a user needs to change or cancel his/her appointment, he/she must Edit/Delete it on the analytical instrument calendar on www.schedulebook.com.
Users must contact Core staff directly to change or cancel a cell sorting or assisted analysis appointment. However, the time may be charged entirely or partially depending on how the reduced/canceled time is used by others.
If a user fails to show up for his/her scheduled appointment, he/she will be charged for the time scheduled and will be reported as a Minor Violation (see violations and fines at the end of this page. Unused time will be charged to non NIH funds (02 or 04 accounts).
Last User of the Day
It is the responsibility of the last user of the day to turn off the analytical instrument. If the last person of the day cannot make his/her scheduled time, he/she must inform the person before so that that person knows to turn off the machine.
It is the user’s responsibility to export the experiment data from the analytical instrument to the hard drive, and subsequently to his/her own device or server.
To ensure that the facility has an extra copy of the data and prevent unexpected data loss, we strongly recommend that users do not delete their exported experiments from the hard drive. However, users must delete their experiment from FACSDiva software after exporting it.
The facility will have a routine schedule (monthly) for data backup.
Guidelines for Using Analyzer (updated 4/19/2018)
Instrument preparation
During the workday, the instrument should be QC by Core staff every morning before service.
The user during the day must fill the sheath to the top line of the sheath tank, empty the waste tank if it is ½ full, add the bleach to the bottom line of the waste tank, and remove any air bubbles in the sheath filter.
The first user before QC must follow ALL the steps of start-up procedure posted on or next to the instrument.
Using instruments during weekends, university breaks and holidays
Users must schedule experiments on the calendar online.
Users are responsible for shutting the instrument down after use .A user should only leave the instrument on if he/she has talked to the person scheduled after them and know that he/she will be using the instrument. If a user needs the person scheduled before to leave the machine on, he/she must talk to that person.
Please follow ALL the steps of start-up and shut-down procedures posted on or next to the instruments. To avoid a clogging issue, cells must be filtered prior to running your sample.
Sample requirements
Samples must be in the single-cell suspension. Cells must be filtered (<80um) at the machine just prior to runinng your sample. The user will be charged for the time used to unclog the instrument.
· Basic Filtering Procedure:
1. Mix cells well and take them with pipette
2. Hold the mesh over the tube -- 80um Nylon mesh (supplied by Flow Core with approximately 2cm x2cm cuts) (http://www.elkofiltering.com/store/p/434-Nylon-Mesh-80-Micron-Open-Area-37-Width-40-in.aspx)
3. Place pipette tip in the center of the tube opening over the mesh and inject cells through mesh as quick as possible. If necessary, wash the mesh with small amount of dilute solution
· Notes:
1. Universal precautions are to be followed according to the EH&S guideline (http://www.ehs.pitt.edu/biological/ ) when filtering the potential bio-hazards or infectious cells without fixation in BSL2 lab at SBST Rm756
2. This protocol can recover 185ul from original 200ul by using P200 pipette
3. To avoid introducing bubble into the system, minimal 300ul of sample is recommended when using standard 12 x 75mm round-bottom tubes. Minimum volume for the Aurora is 100ul of sample.
4. If the samples stained in the 96-well plate and the user would prefer to use multichannel pipettes to filter into another plate, the user can bring their own mesh for filtering
The sample must be in a 12 x 75mm Polystyrene tube (Falcon #s 352052, 352054, or 352058) for the LSR II and Fortessa. For the Aurora, samples can be in 12x75mm Polystrene or Polypropylene tubes. Transfer of samples may be required if using inappropriate tubes.
Minimum volume of each sample is 0.3ml on the LSR II and Fortessa. Minimum volume for the Aurora is 0.1ml.
Samples containing potential bio-hazards are recommended to be fixed in a 0.5-2% paraformaldehyde solution for at least 30 minutes before acquisition. If the user has to run those samples without fixation, he/she must follow the special “Guidelines for running potential bio-hazardous specimen without fixation on STI Fortessa in BST Room S756” or running them on the Aurora by folowing the BSL2 requirements from EH&S. Samples containing known biohazard or infectious agents which are ABOVE BSL2 MUST be fixed in a 0.5%-2% paraformaldehyde solution for at least 30 minutes before acquistion.
Users cannot discard tubes with cells in fixative solution (e.g. paraformaldehyde) in the Core. All tubes with unfixed human, non-human primate cells or virally-transduced or infected cells must be disposed in your own laboratory by following EH&S recommendations.
No radioactive materials are allowed in the facility.
Cleaning after Acquisition
The “Cleaning and shut-down procedure” has been posted on or next to the instrument. Users must clean the instrument after finishing the experiment by following the outlined cleaning procedure.
The last user of the day must shut down the instrument after the cleaning procedure.
Running potential bio-hazardous specimen without fixation on STI Fortessa in BST Rm S756
It has been observed that the aerosol can be generated when taking the sample tube off from the sample injection port (SIP). Since some users may run potential bio-hazardous specimen without fixation, for the purpose of protecting operator as well as other users, this shared instrument is now located inside a class II biosafety cabinet in a Biosafety Level 2 (BSL2) certified room. By following the BSL2 requirements from EH&S, we request that samples containing known biohazard or infectious agents which are ABOVE BSL2 MUST be fixed in a 0.5-2% paraformaldehyde solution for at least 30 minutes before acquisition. If a user has to run a potentially bio-hazardous specimen without fixation, such as unfixed human, non-human primate cells, or virally-transduced or infected cells, in addition to following our general guidelines for using LSR II and Fortessa, he/she must also do the following:
1. Before starting the experiment, users need to TURN ON the hood
2. After finishing the experiment, the user MUST decontaminate the working area by spraying Viraguard around and under the SIP areas and let it stay for at least 10 min. Then follow the “cleaning and shut down procedure” by running 10% Bleach tube for 10 min instead of 5 min. Before the user leaves the lab, he/she must wipe the areas that have been sprayed with Viraguard by using a paper towel, and then he/she must TURN OFF the hood.
Changing optical filter(s) for special application (updated on 11/17/2015)
If the default configuration is not optimal for the special application, the user should contact Core staff for assistance. If the Core is unable to provide/purchase the filter(s) for the user's special application, the user is then allowed to purchase or bring the filter(s) on his/her own expense. However, no matter who owns the filter(s), only Core staff and trained users can change and restore the filters on the instruments during the work hours. During off-work hours (nights, weekends or holidays), only trained users are allowed to change and restore the filter(s). The activities must be recorded on the log sheet next to the instrument. The user should schedule the experiment with the special application accordingly. You will be held responsible for costs assosiated with not returning the filters to the standard configuration.
Problem handling and reporting
Please fill out the on-line problem log form (the home page of Internet Explorer on each cytometer). An e-mail will be automaticall sent to the Core staff. Staff will respond as necessary. Flow Staff emergency contacts are posted on or next to the instrument.
If the user experiences an instrumental or technical problem and he/she or a colleague manages to fix it, he/she must still describe the situation in the on-line problem log - this way, there is a record to track recurring problems.
If the user experiences an instrumental or technical problem that he/she or the colleague is unable to handle and needs immediate assistance, he/she should contact Core staff through phone (office or cell) to get direct instructions. If the problem is not urgent, he/she should notify Core staff by recording it in the on-line problem log.
If the user experiences the general emergency situation, such as fire, chemical, biological spill or personal injury in the Core, he/she must follow the EH&S guideline, which every research person is already trained to do before using the Core. The “Laboratory Emergency Response Guide” from EH&S is posted on the wall for the user’s convenience.
Guidelines for Using High-Speed Cell Sorter
Sample Requirements
Cells (if <40 μm) in all samples and controls must be filtered through a <40 μm nylon mesh filter to prevent clogging of the instrument right before they are brought into the sorting room or right before they are introduced into the cell sorter even if the cells have been filtered earlier before staining in the user’s lab.
Samples can be in any of the following tubes: 12 x 75mm, 15ml, or 1ml.
The concentration of lymphocyte-like cells can be up to 5 x 107 cells/ml. The first time user always brings extra sample solution in case it is needed for sample dilution.
For a multicolor experiment, the user must bring an unstained control tube and a single-color stained tube (ideally >5% positive) if the compensation is required for the “color” used. The user should consult Core staff for the controls required for the instrument set up if there is confusion.
No radioactive materials are allowed in the facility.
Unfixed samples known to contain BSL-3 pathogens or unfixed samples recommended to sort at BSL3 will not be accepted. ( See RBL sorting below) The Operational Director will make the decision concerning sorting of other known infectious agents on an individual basis in consultation with the EH&S Biosafety Officer when necessary.
Criteria for samples containing potential bio-hazards, such as unfixed human, non-human primate, virally-transduced or infected cells:
1. Sample must be contained in a leak-proof container with a secure lid and be clearly labeled with a sample identifier and a biohazard symbol, if needed.
2. Sample must be clump free and in an appropriate tube for sorting (12 x 75 snap cap tube or 15 ml tube with cap).
3. Sample must not contaminate the outside of the tube.
Collection Container
Users need to bring a collection container which can be any of the following options:
1. Tubes: 12 x 75mm, 15ml, or 1ml. For 4-way sorting, only 12 x 75mm tubes can be used.
2. Plates: 24-, 96- (including PCR plate), 384-well, or custom size
3. Trays/dishes or slides
The collection container should be filled with the desired volume of collection medium.
Regional Biocontainment Laboratory: FACS Aria access (NEW 7/20/2020)
Given the reorganization of the support staff in CVR and following a review of flow cytometry provision the center will no longer directly maintain the FACS Aria BSL-3. This is an instrument which is used very infrequently and has failed to draw a sufficiently large cohort of researchers to warrant the significant investment required to maintain it year on year.
CVR and the Immunology Flow Core have entered into an agreement to keep the FACS Aria BSL-3 operational for as long as possible. However, there are no current plans to replace the machine if it fails. The following conditions apply to use:
- Tim Sturgeon (Senior Flow Cytometry Specialist) will run all samples and his time can be booked by contacting sturgeon@pitt.edu
- CVR will charge $40/hour of usage time to cover the cost of the RBL access, PPE, facility maintenance, RBL administrative costs etc.
- Hourly fees for cell sorting, separate from the RBL fees, are listed here: https://www.immunology.pitt.edu/core-facilities/flow-cytometry/fees
- Upon exiting the RBL Tim Sturgeon (flow core) will notify Mike Mehalic (CVR) of RBL usage time. Segments of an hour will be billed for a full hour.
- Prior to use of the equipment a pre-experiment 2 hour “equipment ready” entry must be performed to ensure the machine remains in working order. If this is not the case no experiments can be planned and CVR and Unified Flow Core leadership will determine if the machine has reached the ended of its operational lifespan.
- Equipment ready” certification must be performed every week if experiments run over 5 working days/1 calendar week.The user is responsible for CVR entry fees and flow technician time associated with this instrument certification prior to the experiment.
- If the machine is in working order, as certified by Tim Sturgeon, a booking can be made with the Flow Core to have BSL-3 samples sorted.
- Users must supply all consumables required (as directed by Tim Sturgeon).
- CVR is not responsible for the loss of data or the breakdown of the FACS Aria during the course of an experimental run.
Guidelines for using ImageStream
Instrument preparation
During the work day, the fluidic Startup and ASSIST should be done by Core staff every morning before service.
The user should check to be sure that the buffer containers are full and the waste tank is empty.
The first user before Core staff starts the instrument must follow ALL the steps of start-up procedure posted on or next to the instrument.
Sample requirements
Samples must be in single-cell suspension. If the user experiences significant sample clumping, cells need to be filtered through 80um (or less) Nylon mesh (Small Parts, Inc. cat# CMN-0074).
For the manual acquisition, the sample must be in a 1.5ml microcentrifuge tube (siliconized, Sigma, cat# T4816). For auto-sampler, the samples must be in the round-bottom 96-well plate (Corning, cat#3790). The pierceable cover (X-Pierce, cat# XP-100) is recommended for the plate.
Cells should be in 1 x PBS with no more than 2% FBS at the concentration of 1 million/50ul. Minimum volume of each sample is 20ul.
Samples containing known biohazard or infectious agents which are ABOVE BSL2 MUST be fixed in a 0.5-2% paraformaldehyde solution for at least 30 minutes before acquisition. If a user has to run a potentially bio-hazardous specimen without fixation, such as unfixed human, non-human primate cells, or virally-transduced or infected cells, he/she must follow the BSL2 requirements from EH&S. In addition to that, the user is required to clean the system by loading and running a tube of 100% Bleach (~ 200ul) for 10 min and then a tube of 1 x PBS for about 5 min after finishing the experiment.
No radioactive materials are allowed in the facility
Useful links from Amnis/Millipore:
Filtering samples; Sample Prep Guide; Data acquisition form
Recording the time used
For billing purposes, it is necessary for the user to record the time on the log sheet when starting and finishing an experiment.
Using instruments during weekends, university breaks and holidays
The user should follow the procedures under the “Guidelines for using analyzer”
Problem handling and reporting
The user should follow the procedures under the “Guidelines for using analyzer”
Using the flow core is a privilege with responsibilities. Our researchers depend on each other to ensure that core policies are followed for instrument operations, scheduling, and cleaning. The core will enforce policies as follows:
Minor Violations:
Not filling the sheath.
Not emptying the waste.
Not recording the clean experiment after use.
Violating sign up rules including logging in under a name that is not your own or giving another researcher permission to use your username and password.
Not filtering samples at the cytometer.
Not showing up for a scheduled appointment on the cytometer or sorter.
First offense: E-mail warning to user and PI.
Second offense: 1 hour usage penalty.
Third offense: 2 hour usage penalty and suspension of user privileges until mandatory retraining requirement is completed.
Fourth offense: suspension of facility privileges. At the discretion of the Core Director (Borghesi) restoration of privileges may be discussed at an in person meeting with the user, PI, and Core Director.
Major Violations:
Leaving the machine in a state non-compliant with our policies such that it cannot be used without reporting in the problem log.
Leaving the Cytometer on overnight.
Leaving the cytometer in run.
Not returning the filter set-up to the default configuration as posted near cytometer.
First offense: 1 hour usage penalty and e-mail to user, PI and Core Director
Second offense: 2 hour penalty and suspension of user privileges until mandatory retraining requirement is completed.
Third offense: Removal of privileges. At the discretion of the Core Director (Borghesi) restoration of privileges may be discussed at an in person meeting with the user, PI, and Core Director.