UFC Policies
NIH instrumentation proposals must list publications that have relied on equipment previously purchased using such support. This utilization must be acknowledged in the publications. If your studies directly or indirectly benefitted from the below instruments, please acknowledge the corresponding NIH award when submitting your manuscript for publication.
Suggested wording: “This work benefitted from (instrument from list) funded by NIH (grant number from list).”
Cytek Aurora CS Spectral Sorter
S10OD032265
PI: Kane
Canopy Biosciences Chip Cytometer
S10OD030396
PI: Kane
For Grant submissions copy the following statement:
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 II (5 laser, 18 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.
Scheduling - Sorters
To book time on a sorter, a user must have first created an iLab account and linked it with their PI (to set up the billing information).
To schedule a sort at the BST or Assembly Building:
- Log into iLab (most of you will use your Pitt credentials in SSO).
- Click on "Schedule Equipment" tab, go to either BST or Assembly sorters, and click on the name of the one you want.
- Choose a block of time, and fill out the requested information, including the sort request form that appears. This gives us valuable information about your experiment so we can have the sorter ready for you when you arrive.
- Please do this at least 24 hours (not including the weekend!) before the sort is needed. We cannot guarantee availability of a sorter for a same day sort even if the sorter is unscheduled for that time, due to staff availability: training schedules, which building we are located at at the time, sorter or other machine QC/warmups/cleanings, etc.
- You will receive a confirmation email within 1-2 business days. You can also confirm that you scheduled a sort by checking the same calendar in iLab. Approved sort requests will be purple; pending requests will still be orange.
Please refer to Sorting Policies and Guidelines for more information.
Scheduling - Analyzers
- Trained users must schedule experiments on the individual instrument calendar in iLab.
- Please note that you can't sign up for a machine you are not trained on or if you don't have a valid/active grant linked to your iLab account.
- Untrained users can request training on specific types of machines from within iLab by going to "Request Services" and filling out the "Request for Instrument Training/Access" form. You will be contacted by someone from the Flow Core within 1-2 business to schedule a training session.
- Users should try not to overbook time because you are blocking another user from that unused time.
- 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. Therefore, please be sure to add 10-15 min to your estimated experiment time when scheduling an analyzer to allow time for cleaning and data transfer.
- 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, they must inform the person before so that that person knows to turn off the machine.
To analyze samples containing potential bio-hazards (BSL2) without fixation, such as unfixed human, non-human primate or virally-transduced or infected cells, etc., users must schedule using the instrument (Fortessa-7 South) within a containment hood, which is located in south BST Room S756 or use the Aurora in BST room W1005.
Using instruments during weekends, university breaks, and holidays
- Users must schedule experiments in iLab.
- 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 clogging issues, all samples must be filtered prior to running.
Changing or canceling an appointment
In order to maximize instrument accessibility, the cancellation window to avoid fees is 48 hours for sorters and 24 hours for analyzers.
SORT CANCELLATION POLICY (48 HOUR WINDOW)
- Users cancelling >48 hrs prior to sort reservation will not be charged.
- Users cancelling 24-48 hrs prior to sort reservation will be charged for 1 hour of sort time.
- Users cancelling <24 hrs prior to the sort will be charged for 1 hour of sort time.
- Time may be charged entirely or partially depending on how the reduced/canceled time is used by others.
Users must contact Core Staff at flowcore@pitt.edu to change or cancel a cell sorting or assisted analysis appointment.
ANALYZER CANCELLATION POLICY (24 HOUR WINDOW)
- 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 malfunction.
- The time may be charged entirely or partially depending on how the reduced/canceled time is used by others.
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 in iLab.
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.
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 quickly as possible. If necessary, wash the mesh with small amount of dilute solution (e.g. PBS).
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. An e-mail will be automatically 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.
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. 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.
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:
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 (Kane) 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 (Kane) restoration of privileges may be discussed at an in person meeting with the user, PI, and Core Director.