Policies, Requirements and Procedures

 

 
Publications Relying on NIH S10 Equipment
 
Effective 2015, 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 (updated 04/21/17), 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).”
 
SPECIAL BD LSRFORTESSATM
1S10OD011925-01
PI: Borghesi
(this is the IMM Fortessa in BST E1514)
 
IMAGESTREAMX MARKII
1S10OD019942-01
PI: Borghesi
(this is the ImageStream in BST E1005)

 

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 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), and Amnis ImageStream imaging cytometer (6 laser, 10 detector). The core is approved for specialized BSL2+ sorting.

Training

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.

 

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Scheduling and Cancellation

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 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 only schedule using the instrument (STI Fortessa) within a containment hood, which is located in south BST Room S756

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 a day 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.  Repeatedly failing to show up may cause the user’s privileges to be revoked for a month.

 

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. 

 

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Data Backup

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.

 

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 Guidelines for Using Analyzer

 

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, always check the sample tube before running on the instrument and filter it if there is a visible clump.

 

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

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). Transfer of samples may be required if using inappropriate tubes.

Minimum volume of each sample is 0.3ml.

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”

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

The basic trouble shooting binder can be found in the room. Problem log sheets are available on or next to the instrument for users to record the problem. 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 problem log sheet. 

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 on the problem log sheet, email or phone call. 

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.

 

 

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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. 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.

 

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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”

 

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